Simultaneous, integrated selection and evolution of antibody/protein performance and expression in production hosts

ABSTRACT

The present disclosure provides methods of integrating therapeutic protein and antibody generation and/or selection, evolution and expression in a eukaryotic host for manufacturing in a single system. Therapeutic proteins, including antibodies, are generated, optimized and manufactured in the same eukaryotic host system. The disclosed system of Comprehensive Integrated Antibody Optimization (CIAO!™) allows for simultaneous evolution of protein performance and expression optimization.

RELATED APPLICATION DATA

This application is a division of U.S. patent application Ser. nN.16/118,673, filed on Aug. 31, 2018, now U.S. Pat. No. 10.920,216, issuedon Feb. 16, 2021, which, in turn, is a continuation of U.S. patentapplication Ser. No. 13/298,559, flied on Nov. 17, 2011, now U.S. Patentno. 10,106,788 issued on Oct. 23, 2018, which, in turn, is acontinuation-in-part of International Patent Application Ser. No.PCT/US10/42302, filed Jul. 16, 2010, which claims to the benefit of U.S.Provisional Application No: 61/271,168, filed Jul. 17, 2009, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

In a particular aspect, the present invention is relevant to proteinsand to their optimization by protein evolution. Protein therapeutics arediscovered, evolved and manufactured in the same host using the samegenetic systems.

BACKGROUND OF THE INVENTION

A variety of antibody and other protein systems that generate candidateprotein therapeutic molecules have been designed, developed andimplemented. More recently, many evolution systems have been developedto enhance the function of the proteins. Separately, mammalianexpression systems have been developed for high yield production ofantibodies and other proteins for therapeutic applications. To date, nogroup has developed a system to enable the generation, evolution of anantibody or protein, and protein production/manufacturing in a singleefficient mammalian expression system.

Many antibodies are developed using bacteria phage display systems inbacteria, while expression of full-length antibodies is carried outprimarily in mammalian cells. This lack of similarity makes evolution orselection of clones for expression impossible. Additional barriersinclude the traditional requirement for large numbers of variants to bescreened using traditional technologies and high level mammalianexpression systems have been optimized for expression, not cloning oflarge numbers of variants. An integrated antibody/protein selection,evolution and mammalian expression system has not previously beendesigned. The use of surface display in mammalian cells for handlinglarge numbers, combined with non-stochastic evolution of theantibody/protein inside of an optimized mammalian host cell, increasesthe likelihood of success and greatly accelerates the process forgenerating an optimized antibody/protein that will express at highenough levels in mammalian cells desired in manufacturing.

SUMMARY OF THE INVENTION

The present disclosure provides methods of integrating therapeuticprotein (including antibodies) generation and/or selection, evolutionand expression in a eukaryotic host, such as a mammalian cell host or ayeast cell host, for manufacturing in a single system. Therapeuticproteins, including antibodies, are generated, optimized andmanufactured in the same eukaryotic host system. The disclosed system ofComprehensive Integrated Antibody Optimization (CIAO!™) allows forsimultaneous evolution of protein performance and expressionoptimization.

In one embodiment the disclosure provides a method of selection,evolution and expression of an antibody in a mammalian cell productionhost; the method comprising evolving a template antibody from an(anti-antibody) library to produce a set of mutant antibodies in theeukaryotic cell production host with antibody cell surface display;screening the mutant antibodies for the at least one predeterminedproperty, characteristic or activity; and expressing an up-mutantantibody in the same mammalian cell production host as used in thegenerating step. In one aspect, the antigen is pre-selected. In anotheraspect, the anti-antigen antibody library is a humanized anti-antigenantibody library. In some aspects, the method includes a step ofscreening an anti-antigen antibody library generated from a eukaryoticcell production host with antibody cell surface display for at least onepredetermined property, characteristic or activity,

In one aspect, the mammalian cell production host is selected from 3T3mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, dogepithelial cells; Hela human epithelial cells; PtK1 rat kangarooepithelial cells; SP2/0 mouse plasma cells; and NS0 mouse mouse plasmacells; HEK 293 human embryonic kidney cells; COS monkey kidney cells;CHO, CHO-S Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1mouse embryonic cells; H1 human embryonic cells; H9 human embryoniccells; PER C.6, human embryonic cells; S. cerevisiae yeast cells; orpicchia yeast cells. In a particular aspect, the mammalian system isCHO-S or HEK293. In one aspect, the screening steps utilizefluorescence-activated cell sorting (FACS).

In another aspect, the evolving step comprises producing a set of mutantantibodies formed from the template antibody having m complementarydetermining regions (CDR), wherein m is an integer selected from 1, 2,3, 4, 5 or 6, each said CDR comprising n amino acid residues, the methodcomprising generating m×n separate sets of antibodies, each setcomprising member antibodies having X number of different predeterminedamino acid residues at a single predetermined position of the CDR;wherein each set of antibodies differs in the single predeterminedposition; and the number of different member antibodies generated isequivalent to m×n×X. In a particular aspect, m is 6.

In one aspect, the evolving step comprises generating n-1 separate setsof mutant polypeptides from the template antibody, each set comprisingmember polypeptides having X number of different predetermined aminoacid residues at a single predetermined position of the polypeptide;wherein each set of polypeptides differs in the single predeterminedposition; and the number of different member polypeptides generated isequivalent to [n-1]×X. In one aspect, X represents the 19 naturallyoccurring amino acid residues not present in a given position of thetemplate polypeptide.

In another aspect, the screening comprises assaying each memberpolypeptide for at least one predetermined property, characteristic oractivity; identifying any change in said property, characteristic oractivity of the member polypeptide relative to the template polypeptide;creating a functional map wherein the functional map is used to identifypositions and mutations in the mutant polypeptide which result in anup-mutant and/or a silent mutation compared to the template polypeptide.

In other aspects, screening the mutant antibodies for at least onepredetermined property, characteristic or activity in the methodsdisclosed herein is done under screening conditions that mimicformulation conditions. The conditions that mimic manufacturing orformulation conditions may include screening at a pH of about 4.5 toabout 6.0. The pH may be adjusted using a sale of an acid selected fromthe group consisting of acetate, succinate, gluconate, hisitidine,citrate. Such screening conditions may utilize a polyol, such as sucroseor trehalose in the range of 1 to 15%. The screening conditions mayutilize a surfactant, such as a polysorbate or poloxamer at aconcentration of 0.001%-0.5%, 0.005% to 0.2% or 01% to 0.1%. Thescreening conditions may also utilize a carrier or stabilize, such as anantioxidant, including ascorbic acid or methionine. Such screeningconditions may also utilize a chelating agent, such as EDTA. Thescreening conditions may utilize a metal complex, such as a Zn-proteincomplex. The screening conditions may utilize a polymer, such as apolyester. The screening conditions utilize may an additional salt, suchas a sodium salt, a calcium salt a zinc salt and combinations of thesesalts. The screening may utilize a step of freeze-drying the mutantantibodies, of precipitating the mutant antibodies or both.

In other aspects, the eukaryotic cell production host utilized in themethods disclosed herein incorporates an unnatural amino acid into themutant antibodies. The unnatural amino acid may contain a reactivegroup, such as an azide moiety or alkynyl moiety. Examples of unnaturalamino acids include an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine,a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a4-propyl-L-tyrosine, a tri-O-acetyl-G1cNAcβ-serine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine, and an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or any combination thereof; an amino acidwith a photoactivatable cross-linker; a spin-labeled amino acid; afluorescent amino acid; an amino acid with a novel functional group; anamino acid that covalently or noncovalently interacts with anothermolecule; a metal binding amino acid; a metal-containing amino acid; aradioactive amino acid; a photocaged amino acid; a photoisomerizableamino acid; a biotin or biotin-analogue containing amino acid; aglycosylated or carbohydrate modified amino acid; a keto containingamino acid; an amino acid comprising polyethylene glycol; an amino acidcomprising polyether; a heavy atom substituted amino acid; a chemicallycleavable or photocleavable amino acid; an amino acid with an elongatedside chain; an amino acid containing a toxic group; a sugar substitutedamino acid, e.g., a sugar substituted serine or the like; acarbon-linked sugar-containing amino acid; a redox-active amino acid; anα-hydroxy containing acid; an amino thio acid containing amino acid; anα,αdisubstituted amino acid; a β-amino acid; and a cyclic amino acidother than proline.

In other aspects, the eukaryotic cell production host utilized in themethods described herein reduces fucose content in the mutantantibodies. In some aspect the fucose content is eliminated. In someaspects, the eukaryotic cell production host lacks a fucosyltransferaseenzyme. In other aspects, the eukaryotic cell production host may beMs704, Ms705, and Ms709.

In other aspects, screening step(s) utilized in the methods describedherein include screening to eliminate antibodies exhibiting anundesirable immune response.

In another aspect, the functional map is used to identify one or more ofthe group consisting of (a) positions and mutations which do not affectthe activity of the mutant polypeptide compared to the templatepolypeptide; (b) fully mutable sites compared to the templatepolypeptide; and (c) positions and mutations which result in anup-mutant compared to the template polypeptide.

In another aspect, the antibody fragment is selected from a heavy chain,light chain, variable domain, constant domain, hypervariable region,complementarity determining region 1 (CDR1), complementarity determiningregion 2 (CDR2), and complementarity determining region 3 (CDR3).

In another aspect, the generating step comprises subjecting acodon-containing polynucleotide encoding for said template polypeptideto polymerase-based amplification using a 64-fold degenerateoligonucleotide for each codon to be mutagenized, wherein each of said64-fold degenerate oligonucleotides is comprised of a first homologoussequence and a degenerate N,N,N triplet sequence, so as to generate aset of progeny polynucleotides; and subjecting said set of progenypolynucleotides to clonal amplification such that polypeptides encodedby the progeny polynucleotides are expressed.

In another aspect, the predetermined property, characteristic oractivity is selected from reduction of protein-protein aggregation,enhancement of protein stability, increased protein solubility,introduction of glycosylation sites, introduction of conjugation sites,reduction of immunogenicity, enhancement of protein expression, increasein antigen affinity, decrease in antigen affinity, change in bindingaffinity, change in immunogenicity, or enhancement of specificity.

In another embodiment, the disclosure provides a method of evolution andexpression of an antibody in a mammalian cell production host; themethod comprising selecting a template antibody; evolving the templateantibody to produce a set of mutant antibodies in a mammalian cellproduction host with antibody cell surface display; screening the mutantantibodies for the at least one predetermined property, characteristicor activity; selecting an up-mutant antibody from the set of mutantantibodies based upon optimization of the at least one predeterminedproperty, characteristic or activity compared to the template antibody;and expressing the up-mutant antibody in the same mammalian cellproduction host used in the evolving step.

In one aspect, the screening step comprises creating a functional mapwherein the functional map is used to identify positions and mutationsin the mutant polypeptide which result in an up-mutant and/or a silentmutation compared to the template polypeptide. In another aspect, thescreening steps comprise fluorescence-activated cell sorting (FACS).

In one aspect, the mammalian cell production host is selected from 3T3mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, dogepithelial cells; Hela human epithelial cells; PtK1 rat kangarooepithelial cells; SP2/0 mouse plasma cells; and NS0 mouse mouse plasmacells; HEK 293 human embryonic kidney cells; COS monkey kidney cells;CHO, CHO-S Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1mouse embryonic cells; H1 human embryonic cells; H9 human embryoniccells; and PER C.6, human embryonic cells. In a particular aspect, themammalian system is CHO-S or HEK293. In another aspect, the cellproduction host is selected from CHOK1SV or NSO host cell lines.

In another embodiment, the disclosure provides a method of evolution andexpression of a protein in a eukaryotic cell production host; the methodcomprising selecting a template antibody; evolving the template antibodyto produce a set of mutant antibodies in a eukaryotic cell productionhost; screening the mutant antibodies for at least one predeterminedproperty, characteristic or activity; selecting an up-mutant antibodyfrom the set of mutant antibodies based upon optimization of the atleast one predetermined property, characteristic or activity compared tothe template antibody; and expressing the up-mutant antibody in the sameeukaryotic cell production host as in the evolving step for anycommercial scale.

In one embodiment, the disclosure provides a method of evolution andmanufacturing of a human protein in a cell production host; the methodcomprising generating a human protein library in a cell production hostselected from one of the group consisting of a bacterial or eukaryoticproduction host; screening the library for at least one predeterminedproperty, characteristic or activity; selecting a template human proteinfrom the library based upon the at least one predetermined property,characteristic or activity; evolving the template human protein toproduce a set of mutant human proteins in the cell production host;screening the set of mutant human proteins for the at least onepredetermined property, characteristic or activity and screening formodified expression; selecting an up-mutant human protein from the setof mutant human proteins based upon (1) optimization of the at least onepredetermined property, characteristic or activity and (2) modifiedexpression when compared to the template human protein; andmanufacturing the human protein comprising expressing the up-mutanthuman protein in the same production host as in the generating step. Inone aspect, the modified expression in the selecting step is improvedexpression.

In another aspect, the evolving step comprises an evolution techniqueselected from one of comprehensive positional evolution (CPE);comprehensive positional insertion evolution (CPI); comprehensivepositional deletion evolution (CPD); comprehensive positional evolution(CPE) followed by combinatorial protein synthesis (CPS); comprehensivepositional deletion evolution (CPD) followed by combinatorial proteinsynthesis (CPS); or comprehensive positional deletion evolution (CPD)followed by combinatorial protein synthesis (CPS).

In one aspect, the human protein is an antibody. In another aspect, theantibody is a full length antibody.

In another aspect, the at least one predetermined property,characteristic or activity in screening step (e) comprises one or moreof (1) screening for a silent mutation and (2) screening for a missensemutation; compared to the template antibody.

In another aspect, one or more portions of the antibody selected from Fcand Fv; framework; and one or more CDRs are modified in the up-mutanthuman antibody compared to the template human antibody is evolved.

In another aspect, the screening step comprises screening the set ofmutant human proteins for the at least one predetermined property,characteristic or activity and screening for modified expressionsimultaneously.

In a further aspect, the human protein for evolution and manufacturingis selected from an enzyme cytokine, receptor, DNA binding protein,chelating agent or hormone.

In another aspect, the cell production host is a eukaryotic productionhost and the evolving step comprises evolving the template human proteinto produce a set of mutant human proteins in the eukaryotic cellproduction host with cell surface display.

In another embodiment, the disclosure provides a method of evolution forenhanced expression and manufacturing of a human protein in a eukaryoticcell production host; the method comprising selecting a template humanprotein for evolution; evolving the template human protein comprisinggeneration of mutant codons encoding the template human protein toproduce a set of mutant human proteins in the production host; screeningthe set of mutant human proteins for at least one predeterminedproperty, characteristic or activity and screening for enhancedexpression when compared to the template human protein; selecting anup-mutant human protein from the set of mutant human proteins based upon(1) retention or optimization of the at least one predeterminedproperty, characteristic or activity and (2) enhanced expression whencompared to the template human protein; and manufacturing the up-mutanthuman protein comprising expressing the up-mutant human protein in thesame production host as in the evolving step.

In one aspect, the mutant codons of the up-mutant human protein resultin at least one silent mutation and/or missense mutation. In anotheraspect, the mutant codons of the up-mutant human protein result in atleast one silent mutation.

In a further aspect, the template human protein is an approved ethicalprotein therapeutic drug, and the up-mutant human protein is abiosimilar.

In another aspect, the selecting step comprises selecting an up-mutanthuman protein from the set of mutant human proteins based upon (1)optimization of the at least one predetermined property, characteristicor activity and (2) enhanced expression when compared to the templatehuman protein.

In another embodiment, the disclosure provides a method of identifyingand producing a target human protein, the method comprising generatingan human protein library in a eukaryotic cell production host withprotein cell surface display; screening the library for at least onepredetermined property, characteristic or activity; identifying a targethuman protein from the library based upon the at least one predeterminedproperty, characteristic or activity; and expressing the target humanprotein in the same eukaryotic cell production host as in the generatingstep to produce a target human protein. In one aspect, the target humanprotein is an antibody. In another aspect, the antibody is a full lengthantibody.

In another embodiment, the disclosure provides a method of evolution ofa human protein in a manufacturing host, the method comprising mutatinga template human protein to produce a set of mutant human proteins in amanufacturing host; and screening the set of mutant progeny proteins forat least one predetermined property, characteristic or activity. In oneaspect, the method further comprises selecting an up-mutant humanprotein from the set of mutant human proteins based upon the at leastone predetermined property, characteristic or activity. In anotheraspect, the method further comprises manufacturing the up-mutant humanprotein comprising expressing the up-mutant human protein in the sameproduction host as in the mutating step. In another aspect, theselecting step further comprises selecting an up-mutant human proteinfrom the set of mutant human proteins based upon (1) optimization of theat least one predetermined property, characteristic or activity whencompared to the template human protein, and (2) modified expression whencompared to the template human protein. In one aspect, the modifiedexpression is enhanced expression.

In another embodiment, the disclosure provides a method of evolution andmanufacturing of a human protein in a cell manufacturing host; themethod comprising mutating a template human protein to produce a set ofmutant human proteins in a manufacturing host; screening the set ofmutant human proteins for at least one predetermined property,characteristic or activity and screening for modified expression;

selecting an up-mutant human protein from the set of mutant humanproteins based upon (1) optimization of the at least one predeterminedproperty, characteristic or activity, and (2) modified expression whencompared to the template human protein; and manufacturing the humanprotein comprising expressing the up-mutant human protein in the samemanufacturing host as in the mutating step.

In a further embodiment, the disclosure provides a method of evolutionfor enhanced expression and manufacturing of a human protein in aeukaryotic cell production host; the method comprising mutating atemplate human protein comprising generation of mutant codons encodingthe template human protein to produce a set of mutant human proteins ina manufacturing host; screening the set of mutant human proteins for atleast one predetermined property, characteristic or activity andscreening for enhanced expression when compared to the template humanprotein; selecting an up-mutant human protein from the set of mutanthuman proteins based upon (1) retention or optimization of the at leastone predetermined property, characteristic or activity and (2) enhancedexpression when compared to the template human protein; andmanufacturing the up-mutant human protein in the same manufacturing hostas in the mutating step.

In one aspect, the screening step comprises screening the set of mutanthuman proteins for the at least one predetermined property,characteristic or activity and screening for enhanced expressionsimultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of the CIAO!™ method of integratingtherapeutic protein (e.g., antibody) generation and/or selection,evolution and expression in a eukaryotic host, such as a mammalian cellhost or a yeast cell host, for manufacturing in a single system.

FIG. 2 illustrates how comprehensive positional evolution (CPE™) is usedto generate a molecule specific database (EvoMap™).

FIG. 3 shows a schematic of Comprehensive Positional Synthesis (CPS™)which can be used to combine up-mutants from CPE™.

FIG. 4 shows a schematic of Comprehensive Positional Insertion (CPI™)evolution.

FIG. 5 illustrates one combination of evolution methods: a lengthenednucleic acid from CPI™ evolution is subjected to ComprehensivePositional Evolution (CPE™) and used to generate a molecule specificdatabase (EvoMap™).

FIG. 6 shows a schematic of Comprehensive Positional Deletion (CPD™)evolution.

FIG. 7 illustrates another combination of evolution methods: a shortenednucleic acid from CPD™ evolution is subjected to ComprehensivePositional Evolution (CPE™) and used to generate a molecule specificdatabase (EvoMap™).

DEFINITION OF TERMS

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be described.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

The term “agent” is used herein to denote a polypeptide, a mixture ofpolypeptides, an array of spatially localized compounds (e.g., a VLSIPSpeptide array, polynucleotide array, and/or combinatorial small moleculearray), biological macromolecule, a bacteriophage peptide displaylibrary, a bacteriophage antibody (e.g., scFv) display library, apolysome peptide display library, or an extract made form biologicalmaterials such as bacteria, plants, fungi, or animal (particularmammalian) cells or tissues. Agents are evaluated for potential activityas anti-neoplastics, anti-inflamnmatories or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which does notsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

The term “aggregation” refers to protein self-association, andencompasses any type of interaction or characteristic of proteins.Aggregates can be soluble or covalent, reversible or no-reversible.

“amino acid” as used herein refers to any organic compound that containsan amino group (—NH₂) and a carboxyl group (—COOH); preferably either asfree groups or alternatively after condensation as part of peptidebonds. The “twenty naturally encoded polypeptide-forming alpha-aminoacids” are understood in the art and refer to: alanine (ala or A),arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D),cysteine (cys or C), gluatamic acid (glu or E), glutamine (gln or Q),glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine(leu or L), lysine (lys or K), methionine (met or M), phenylalanine (pheor F), proline (pro or P), serine (ser or S), threonine (thr or T),tryptophan (trp or W), tyrosine (tyr or Y), and valine (val or V).

The term “amplification” means that the number of copies of apolynucleotide is increased.

The term “antibody”, as used herein, refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)2, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen. These antibody fragments, which retain someability to selectively bind to an antigen (e.g., a polypeptide antigen)of the antibody from which they are derived, can be made using wellknown methods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows. Antibodies can be used to isolatepreparative quantities of the antigen by immunoaffinity chromatography.Various other uses of such antibodies are to diagnose and/or stagedisease (e.g., neoplasia) and for therapeutic application to treatdisease, such as for example: neoplasia, autoimmune disease, AIDS,cardiovascular disease, infections, and the like. Chimeric, human-like,humanized or fully human antibodies are particularly useful foradministration to human patients.

An Fab fragment consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

An Fab′ fragment of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner

An (Fab′)2 fragment of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together bytwo disulfide bonds.

An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

A single chain antibody (“SCA”) is a genetically engineered single chainmolecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide liner.

As used herein, the terms “antibody that lacks fucose residues” and“defucosylated antibody” are used interchangeably and are intended torefer to an antibody in which the carbohydrate portion of the antibodydoes not contain a fucosyl residue or from which the fucosyl residue hasbeen removed. An antibody that lacks fucose residues can be generated,for example, by expression of the antibody in a cell or expressionsystem that minimizes or does not attach fucosyl residues to theantibody carbohydrate chain, or by chemical modification of the antibodyto remove fucosyl residues from the carbohydrate chain (e.g. treatmentof the antibody with a fucosidase). As such, the terms “lacks fucoseresidues” and “defucosylated” are not intended to be limited by themechanism by which the antibody with altered carbohydrate structure isprepared.

The term “biosimilar”, also termed “follow-on biologic”, refers toofficially approved new versions of innovator biopharmaceuticalproducts, following patent or exclusivity expiry.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components.Examples of buffers include acetate (e.g. sodium acetate), succinate(such as sodium succinate), gluconate, histidine, citrate and otherorganic acid buffers. Where a freeze-thaw stable formulation is desired,the buffer is preferably not phosphate.

The term “cell production host”, or “manufacturing host”, refers to acell line used for the production or manufacturing of proteins.Eukaryotic cells such as mammalian cells, including, but not limited tohuman, mouse, hamster, rat, monkey cell lines as well as yeast, insectand plant cell lines. Prokaryotic cells can alternatively be utilized.In one aspect, a mammalian cell production host is selected from amember of the group consisting of 3T3 mouse fibroblast cells; BHK21Syrian hamster fibroblast cells; MDCK, dog epithelial cells; Hela humanepithelial cells; PtK1 rat kangaroo epithelial cells; SP2/0 mouse plasmacells; and NS0 mouse mouse plasma cells; HEK 293 human embryonic kidneycells; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells;R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1 humanembryonic cells; H9 human embryonic cells; PER C.6, human embryoniccells. In another aspect, the cell production host is a GS-NS0 orGS-CHOK1 cell line. In another aspect, the cell production host isselected from S. cerevisiae yeast cells; and picchia yeast cells. Inanother aspect, the cell production host is a bacterial cell line.

A molecule that has a “chimeric property” is a molecule that is: 1) inpart homologous and in part heterologous to a first reference molecule;while 2) at the same time being in part homologous and in partheterologous to a second reference molecule; without 3) precluding thepossibility of being at the same time in part homologous and in partheterologous to still one or more additional reference molecules. In anon-limiting embodiment, a chimeric molecule may be prepared byassembling a reassortment of partial molecular sequences. In anon-limiting aspect, a chimeric polynucleotide molecule may be preparedby synthesizing the chimeric polynucleotide using plurality of moleculartemplates, such that the resultant chimeric polynucleotide hasproperties of a plurality of templates.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example,but not limitation, in the human genome the human CD4 gene is thecognate gene to the mouse 3d4 gene, since the sequences and structuresof these two genes indicate that they are highly homologous and bothgenes encode a protein which functions in signaling T cell activationthrough MHC class II-restricted antigen recognition.

The term “commercial scale” means production of a protein or antibody ata scale appropriate for resale.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482 by the homologyalignment algorithm of Needlemen and Wuncsch J. Mol. Biol. 48: 443(1970), by the search of similarity method of Pearson and Lipman Proc.Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

As used herein, the term “complementarity-determining region” and “CDR”refer to the art-recognized term as exemplified by the Kabat andChothia. CDR definitions are also generally known as supervariableregions or hypervariable loops (Chothia and Leks, 1987; Clothia et al.,1989; Kabat et al., 1987; and Tramontano et al., 1990). Variable regiondomains typically comprise the amino-terminal approximately 105-115amino acids of a naturally-occurring immunoglobulin chain (e.g., aminoacids 1-110), although variable domains somewhat shorter or longer arealso suitable for forming single-chain antibodies. The CDRs are parts ofimmunoglobulins that determine the specificity of said molecules andmake contact with a specific ligand. The CDRs are the most variable partof the molecule and contribute to the diversity of these molecules.There are three CDR regions CDR1, CDR2 and CDR3 in each V domain. CDR-Hdepicts a CDR region of a variable heavy chain and CDR-L relates to aCDR region of a variable light chain. H means the variable heavy chainand L means the variable light chain. The CDR regions of an Ig-derivedregion may be determined as described in Kabat (1991). Sequences ofProteins of Immunological Interest, 5th edit., NIH Publication no.91-3242 U.S. Department of Health and Human Services, Chothia (1987) J.Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.

The term “comprehensive” is used herein to refer to a technique ofevolution wherein every possible change is made at each position of atemplate polynucleotide or template polypeptide and the polynucleotideor polypeptide is tested to confirm the intended changes have been made.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “degrading effective” amount refers to the amount of enzymewhich is required to process at least 50% of the substrate, as comparedto substrate not contacted with the enzyme. Preferably, at least 80% ofthe substrate is degraded.

As used herein, the term “defined sequence framework” refers to a set ofdefined sequences that are selected on a non-random basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a (3-sheet structure or may comprise aleucine zipper heptad repeat motif, a zinc-finger domain, among othervariations. A “defined sequence kernal” is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-mer sequence of the 20 conventional amino acids can be any of(20)10 sequences, and (2) a pseudorandom 10-mer sequence of the 20conventional amino acids can be any of (20)10 sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences if each residueposition was allowed to be any of the allowable 20 conventional aminoacids (and/or allowable unconventional amino/imino acids). A definedsequence kernal generally comprises variant and invariant residuepositions and/or comprises variant residue positions which can comprisea residue selected from a defined subset of amino acid residues), andthe like, either segmentally or over the entire length of the individualselected library member sequence. Defined sequence kernels can refer toeither amino acid sequences or polynucleotide sequences. Of illustrationand not limitation, the sequences (NNK)10 and (NNM)10, wherein Nrepresents A, T, G, or C; K represents G or T; and M represents A or C,are defined sequence kernels.

The term “deimmunization” as used herein relates to production of avariant of the template binding molecule, which is modified compared toan original wild type molecule by rendering said variant non-immunogenicor less immunogenic in humans. Deimmunized molecules according to theinvention relate to antibodies or parts thereof (like frameworks and/orCDRs) of non-human origin. Corresponding examples are antibodies orfragments thereof as described in U.S. Pat. No. 4,361,549. The term“deimmunized” also relates to molecules, which show reduced propensityto generate T cell epitopes. In accordance with this invention, the term“reduced propensity to generate T cell epitopes” relates to the removalof T-cell epitopes leading to specific T-cell activation.

Furthermore, reduced propensity to generate T cell epitopes meanssubstitution of amino acids contributing to the formation of T cellepitopes, i.e. substitution of amino acids, which are essential forformation of a T cell epitope. In other words, reduced propensity togenerate T cell epitopes relates to reduced immunogenicity or reducedcapacity to induce antigen independent T cell proliferation. Inaddition, reduced propensity to generate T cell epitopes relates todeimmunization, which means loss or reduction of potential T cellepitopes of amino acid sequences inducing antigen independent T cellproliferation.

The term “T cell epitope” as used herein relates to short peptidesequences which can be released during the degradation of peptides,polypeptide or proteins within cells and subsequently be presented bymolecules of the major histocompatibility complex (MHC) in order totrigger the activation of T cells; see inter alia WO 02/066514. Forpeptides presented by MHC class II such activation of T cells can theninduce an antibody response by direct stimulation of B cells to producesaid antibodies.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a gel to isolate the desired fragment.

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/lox and/or flp/frt systems and the like.DNA shuffling can be random or non-random.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a phytase polypeptide, to which theparatope of an antibody, such as a phytase-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. As used herein “epitope” refers tothat portion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

The term “evolution” refers to a change in at least one property,characteristic or activity of a genetically or synthetically modifiedprotein or antibody when compared to a template protein or antibody.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

A method is provided herein for producing from a template polypeptide aset of progeny polypeptides in which a “full range of single amino acidsubstitutions” is represented at each amino acid position. As usedherein, “full range of single amino acid substitutions” is in referenceto the naturally encoded 20 naturally encoded polypeptide-formingalpha-amino acids, as described herein.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

“Genetic instability”, as used herein, refers to the natural tendency ofhighly repetitive sequences to be lost through a process of reductiveevents generally involving sequence simplification through the loss ofrepeated sequences. Deletions tend to involve the loss of one copy of arepeat and everything between the repeats.

The term “heterologous” means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus, areas of heterology means that areasof polynucleotides or polynucleotides have areas or regions within theirsequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions or areas are for example areas ofmutations.

The term “homologous” or “homeologous” means that one single-strandednucleic acid nucleic acid sequence may hybridize to a complementarysingle-stranded nucleic acid sequence. The degree of hybridization maydepend on a number of factors including the amount of identity betweenthe sequences and the hybridization conditions such as temperature andsalt concentrations as discussed later. Preferably the region ofidentity is greater than about 5 bp, more preferably the region ofidentity is greater than 10 bp.

The term “humanized” is used to describe antibodies whereincomplementarity determining regions (CDRs) from a mammalian animal,e.g., a mouse, are combined with a human framework region. Oftenpolynucleotides encoding the isolated CDRs will be grafted intopolynucleotides encoding a suitable variable region framework (andoptionally constant regions) to form polynucleotides encoding completeantibodies (e.g., humanized or fully-human), antibody fragments, and thelike. In another aspect, besides mouse antibodies, other species can behumanized, such as, for example, other rodent, camel, rabbit, cat, dog,pig, horse, cow, fish, llama and shark. In a broad aspect, any speciesthat produces antibodies can be utilized in the production of humanizedantibodies. Additionally, the antibodies of the invention may bechimeric, human-like, humanized or fully human, in order to reduce theirpotential antigenicity, without reducing their affinity for theirtarget. Chimeric, human-like and humanized antibodies have generallybeen described in the art. By incorporating as little foreign sequenceas possible in the hybrid antibody, the antigenicity is reduced.Preparation of these hybrid antibodies may be carried out by methodswell known in the art.

In an alternative aspect, human or mouse antibodies are adapted to adifferent recipient species, such as an endangered species, in order toprovide therapeutics for the recipient species while protecting themfrom a negative immune response. In this aspect, the frameworks from arecipient species are utilized in combination with CDRs from a known orsecond species antibodies.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest,” Kabat et al., 1987). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. As used herein, a “human framework region” is a frameworkregion that is substantially identical (about 85 or more, usually 90-95or more) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen. In accordance withthis invention, a framework region relates to a region in the V domain(VH or VL domain) of immunoglobulins that provides a protein scaffoldfor the hypervariable complementarity determining regions (CDRs) thatmake contact with the antigen. In each V domain, there are fourframework regions designated FR1, FR2, FR3 and FR4. Framework 1encompasses the region from the N-terminus of the V domain until thebeginning of CDR1, framework 2 relates to the region between CDR1 andCDR2, framework 3 encompasses the region between CDR2 and CDR3 andframework 4 means the region from the end of CDR3 until the C-terminusof the V domain; see, inter alia, Janeway, Immunobiology, GarlandPublishing, 2001, 5th ed. Thus, the framework regions encompass all theregions outside the CDR regions in VH or VL domains.

The person skilled in the art is readily in a position to deduce from agiven sequence the framework regions and, the CDRs; see Kabat (1991)Sequences of Proteins of Immunological Interest, 5th edit., NIHPublication no. 91-3242 U.S. Department of Health and Human Services,Chothia (1987) J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature,342, 877-883.

The benefits of this invention extend to “industrial applications” (orindustrial processes), which term is used to include applications incommercial industry proper (or simply industry) as well asnon-commercial industrial applications (e.g. biomedical research at anon-profit institution). Relevant applications include those in areas ofdiagnosis, medicine, agriculture, manufacturing, and academia.

The term “identical” or “identity” means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, “areas ofidentity” means that regions or areas of a polynucleotide or the overallpolynucleotide are identical or complementary to areas of anotherpolynucleotide or the polynucleotide.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNAmolecule, that is not immediately contiguous with the 5′ and 3′ flankingsequences with which it normally is immediately contiguous when presentin the naturally occurring genome of the organism from which it isderived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e.g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

As used herein “ligand” refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis et al., 1982, p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

The term “mammalian cell surface display” refers to a technique wherebya protein or antibody, or a portion of an antibody, is expressed anddisplayed on a mammalian host cell surface for screening purposes; forexample, by screening for specific antigen binding by a combination ofmagnetic beads and fluorescence-activated cell sorting. In one aspect,mammalian expression vectors are used for simultaneous expression ofimmunoglobulins as both a secreted and cell surface bound form as inDuBridge et al., US 2009/0136950, which is incorporated herein byreference. In another aspect, the techniques of Gao et al. are employedfor a viral vector encoding for a library of antibodies or antibodyfragments are displayed on the cell membranes when expressed in a cellas in Gao et al., US 2007/0111260, incorporated herein by reference.Whole IgG surface display on mammalian cells is known. For example, aAkamatsuu et al. developed a mammalian cell surface display vector,suitable for directly isolating IgG molecules based on theirantigen-binding affinity and biological activity. Using an Epstein-Barrvirus-derived episomal vector, antibody libraries were displayed aswhole IgG molecules on the cell surface and screened for specificantigen binding by a combination of magnetic beads andfluorescence-activated cell sorting. Plasmids encoding antibodies withdesired binding characteristics were recovered from sorted cells andconverted to the form for production of soluble IgG. Akamatsuu et al. J.Immunol. Methods 2007 327(1-2):40-52; incorporated herein by reference.Ho et al. used human embryonic kidney 293T cells that are widely usedfor transient protein expression for cell surface display ofsingle-chain Fv antibodies for affinity maturation. Cells expressing arare mutant antibody with higher affinity were enriched 240-fold by asingle-pass cell sorting from a large excess of cells expressing WTantibody with a slightly lower affinity. Furthermore, a highly enrichedmutant was obtained with increased binding affinity for CD22 after asingle selection of a combinatory library randomizing an intrinsicantibody hotspot. Ho et al. Isolation of anti-CD22 Fv with high affinityby Fv display on human cells, Proc Natl Acad Sci U S A 2006 Jun. 20;103(25): 9637-9642; incorporated herein by reference.

Beerli et al. used B cells specific for an antigen of interest whichwere directly isolated from peripheral blood mononuclear cells (PBMC) ofhuman donors. Recombinant, antigen-specific single-chain Fv (scFv)libraries are generated from this pool of B cells and screened bymammalian cell surface display by using a Sindbis virus expressionsystem. This method allows isolating antigen-specific antibodies by asingle round of FACS. The variable regions (VRs) of the heavy chains(HCs) and light chains (LCs) were isolated from positive clones andrecombinant fully human antibodies produced as whole IgG or Fabfragments. In this manner, several hypermutated high-affinity antibodiesbinding the Qβ virus like particle (VLP), a model viral antigen, as wellas antibodies specific for nicotine were isolated. All antibodies showedhigh expression levels in cell culture. The human nicotine-specific mAbswere validated preclinically in a mouse model. Beerli et al., Isolationof human monoclonal antibodies by mammalian cell display, Proc Natl AcadSci U S A. 2008 Sep. 23; 105(38): 14336-14341; incorporated herein byreference.

Yeast cell surface display is also known, for example, see Kondo andUeda 2004, Yeast cell-surface display-applications of molecular display,Appl. Microbiol. Biotechnol., 64(1): 28-40, which describes for example,a cell-surface engineering system using the yeast Saccharomycescerevisiae. Several representative display systems for the expression inyeast S. cerevisiae are described in Lee et al, 2003, Microbialcell-surface display, TRENDS in Bitechnol. 21(1): 45-52. Also Boder andWittrup 1997, Yeast surface display for screening combinatorialpolypeptide libraries, Nature Biotechnol., 15(6): 553.

The term “manufacturing” refers to production of a protein at asufficient quantity to permit at least Phase I clinical testing of atherapeutic protein, or sufficient quantity for regulatory approval of adiagnostic protein.

The term “missense mutation” refers to a point mutation where a singlenucleotide is changed, resulting in a codon that codes for a differentamino acid. Mutations that change an amino acid to a stop codon arecalled nonsense mutations.

As used herein, a “molecular property to be evolved” includes referenceto molecules comprised of a polynucleotide sequence, molecules comprisedof a polypeptide sequence, and molecules comprised in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include enzymatic activities at specifiedconditions, such as related to temperature; salinity; pressure; pH; andconcentration of glycerol, DMSO, detergent, and/or any other molecularspecies with which contact is made in a reaction environment. Additionalparticularly relevant—but by no means limiting examples of molecularproperties to be evolved include stabilities—e.g., the amount of aresidual molecular property that is present after a specified exposuretime to a specified environment, such as may be encountered duringstorage.

The term “mutating” refers to creating a mutation in a nucleic acidsequence; in the event where the mutation occurs within the codingregion of a protein, it will lead to a codon change which may or may notlead to an amino acid change.

The term “mutations” means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide orpolypeptides. Such mutations may be point mutations such as transitionsor transversions. The mutations may be deletions, insertions orduplications.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

As used herein, the degenerate “N,N,N” nucleotide sequence represents 64possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, a “nucleic acid molecule” is comprised of at least onebase or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a non-radioactive label, is likewise considered a“nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof” or a “nucleotide sequence encoding” a particular protein—as well asother synonymous terms—refer to a DNA sequence which is transcribed andtranslated into an enzyme when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background. However, after the RNA polymerase binds the sequenceand transcription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “nucleic acid encoding an protein” or “DNA encoding anprotein” or “polynucleotide encoding a protein” and other synonymousterms encompasses a polynucleotide which includes only coding sequencefor the protein as well as a polynucleotide which includes additionalcoding and/or non-coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable itsexpressed product.

The instant definition of “assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. Preferablythe adaptors can anneal to PCR primers to facilitate amplification byPCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. Preferably, the collection ofmolecules comprising a library is searchable and separable according toindividual nucleic acid molecule species.

The present invention provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., a phytase polynucleotide) may optionallybe chemically bonded to one or more additional molecular moieties, suchas a vector, or parts of a vector. In a specific—but by no meanslimiting—aspect, a nucleotide construct is exemplified by a DNAexpression DNA expression constructs suitable for the transformation ofa host cell.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated. To achieve polymerase-based amplification (such aswith PCR), a “32-fold degenerate oligonucleotide that is comprised of,in series, at least a first homologous sequence, a degenerate N,N,G/Tsequence, and a second homologous sequence” is mentioned. As used inthis context, “homologous” is in reference to homology between the oligoand the parental polynucleotide that is subjected to thepolymerase-based amplification.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

The term “pharmaceutical formulation” refers to preparations which arein such form as to permit the biological activity of the activeingredients to be effective, and which contain no additional componentswhich are toxic to the subjects to which the formulation would beadministered.

“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg++,Ca++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HC1, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or non-ionic detergents and/or membrane fractionsand/or anti-foam agents and/or scintillants.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. A “reducing sugar” is one which contains a hemiacetal group thatcan reduce metal ions or react covalently with lysine and other aminogroups in proteins and a “nonreducing sugar” is one which does not havethese properties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Nonreducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. Sugar acids include L-gluconate and metallic salts thereof.Where it desired that the formulation is freeze-thaw stable, the polyolis preferably one which does not crystallize at freezing temperatures(e.g. −20° C.) such that it destabilizes the antibody in theformulation. Trehalose is sometimes preferred over sucrose because ofthe superior solution stability of trehalose.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population” means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A “preservative” is a compound which can be included in the formulationto essentially reduce bacterial action therein, thus facilitating theproduction of a multi-use formulation, for example. Examples ofpotential preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g., glycosylation, proteolytic cleavage, dimerizationor oligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modification (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

A “property” can describe any characteristic, including a physical,chemical, or activity characteristic property of a protein or antibodyto be optimized. For example, in certain aspects, the predeterminedproperty, characteristic or activity to be optimized can be selectedfrom is selected from reduction of protein-protein aggregation,enhancement of protein stability, increased protein solubility,increased protein pH stability, increased protein temperature stability,increased protein solvent stability, increased selectivity, decreasedselectivity, introduction of glycosylation sites, introduction ofconjugation sites, reduction of immunogenicity, enhancement of proteinexpression, increase in antigen affinity, decrease in antigen affinity,change in binding affinity, change in immunogenicity, change incatalytic activity, pH optimization, or enhancement of specificity. An“optimized” property refers to a desirable change in a particularproperty in a mutant protein, antibody or cell compared to a templateprotein, antibody or cell, respectively.

As used herein, the term “pseudorandom” refers to a set of sequencesthat have limited variability, such that, for example, the degree ofresidue variability at another position, but any pseudorandom positionis allowed some degree of residue variation, however circumscribed.

“Quasi-repeated units”, as used herein, refers to the repeats to bere-assorted and are by definition not identical. Indeed the method isproposed not only for practically identical encoding units produced bymutagenesis of the identical starting sequence, but also thereassortment of similar or related sequences which may divergesignificantly in some regions. Nevertheless, if the sequences containsufficient homologies to be reasserted by this approach, they can bereferred to as “quasi-repeated” units.

As used herein “random peptide library” refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins contain those random peptides.

As used herein, “random peptide sequence” refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein, “receptor” refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornon-covalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

“Recombinant” proteins refer to proteins produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” proteins are thoseprepared by chemical synthesis.

The term “related polynucleotides” means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

“Reductive reassortment”, as used herein, refers to the increase inmolecular diversity that is accrued through deletion (and/or insertion)events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity,” and“substantial identity.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

“Repetitive Index (RI)”, as used herein, is the average number of copiesof the quasi-repeated units contained in the cloning vector.

An antibody “retains its biological activity” in a pharmaceuticalformulation, if the biological activity of the antibody at a given timeis within about 10% (within the errors of the assay) of the biologicalactivity exhibited at the time the pharmaceutical formulation wasprepared as determined in an antigen binding assay, for example.

The term “saturation” refers to a technique of evolution wherein everypossible change is made at each position of a template polynucleotide ortemplate polypeptide; however the change at each position is notconfirmed by testing, but merely assumed statistically wherein themajority of possible changes or nearly every possible change isestimated to occur at each position of a template.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. This “substantial identity”, as used herein,denotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence having at least 80 percent sequenceidentity, preferably at least 85 percent identity, often 90 to 95percent sequence identity, and most commonly at least 99 percentsequence identity as compared to a reference sequence of a comparisonwindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

The term “silent mutation” refers to a codon change that does not resultin an amino acid change in an expressed polypeptide and is based onredundancy of codon usage for amino acid insertion.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one protein to the sequence of a second protein.Similarity may be determined by procedures which are well-known in theart, for example, a BLAST program (Basic Local Alignment Search Tool atthe National Center for Biological Information).

As used herein, the term “single-chain antibody” refers to a polypeptidecomprising a VH domain and a VL domain in polypeptide linkage, generallyliked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]_(x)) (SEQ ID NO:1), and which may comprise additional amino acid sequences at the amino-and/or carboxy- termini For example, a single-chain antibody maycomprise a tether segment for linking to the encoding polynucleotide. Asan example a scFv is a single-chain antibody. Single-chain antibodiesare generally proteins consisting of one or more polypeptide segments ofat least 10 contiguous amino substantially encoded by genes of theimmunoglobulin superfamily (e.g., see Williams and Barclay, 1989, pp.361-368, which is incorporated herein by reference), most frequentlyencoded by a rodent, non-human primate, avian, porcine bovine, ovine,goat, or human heavy chain or light chain gene sequence. A functionalsingle-chain antibody generally contains a sufficient portion of animmunoglobulin superfamily gene product so as to retain the property ofbinding to a specific target molecule, typically a receptor or antigen(epitope).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Specific hybridization” is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein substantially unrelatedpolynucleotide sequences do not form hybrids in the mixture.

The term “specific polynucleotide” means a polynucleotide having certainend points and having a certain nucleic acid sequence. Twopolynucleotides wherein one polynucleotide has the identical sequence asa portion of the second polynucleotide but different ends comprises twodifferent specific polynucleotides.

A “stable formulation” is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Various analytical techniques formeasuring protein stability are described in various articles in the art, including Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed.,Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones, A. Adv.Drug Delivery Rev. 10: 29-90 (1993), for example. Stability can bemeasured under selected conditions, for example at a selectedtemperature for a selected time period.

“Stringent hybridization conditions” means hybridization will occur onlyif there is at least 90% identity, preferably at least 95% identity andmost preferably at least 97% identity between the sequences. SeeSambrook et al., 1989, which is hereby incorporated by reference in itsentirety.

Also included in the invention are polypeptides having sequences thatare “substantially identical” to the sequence of a polypeptide, such asone of any SEQ ID NO disclosed herein. A “substantially identical” aminoacid sequence is a sequence that differs from a reference sequence onlyby conservative amino acid substitutions, for example, substitutions ofone amino acid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid, or glutamine for asparagine).

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence or by one or morenon-conservative substitutions, deletions, or insertions, particularlywhen such a substitution occurs at a site that is not the active sitethe molecule, and provided that the polypeptide essentially retains itsbehavioural properties. For example, one or more amino acids can bedeleted from a phytase polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for phytase biological activity can beremoved. Such modifications can result in the development of smalleractive phytase polypeptides.

The present invention provides a “substantially pure enzyme”. The term“substantially pure enzyme” is used herein to describe a molecule, suchas a polypeptide (e.g., a phytase polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

As used herein, “template oligopeptide” means a protein for which asecondary library of variants is desired. As will be appreciated bythose in the art, any number of templates find use in the presentinvention. Specifically included within the definition of “proteins” or“oligopeptides” are fragments and domains of known proteins, includingfunctional domains such as enzymatic domains, binding domains, etc., andsmaller fragments, such as turns, loops, etc. That is, portions ofproteins may be used as well. In addition, “protein” as used hereinincludes proteins, oligopeptides and peptides. In addition, proteinvariants, i.e., non-naturally occurring protein analog structures, maybe used.

Suitable proteins include, but are not limited to, industrial andpharmaceutical proteins, including ligands, cell surface receptors,antigens, antibodies, cytokines, hormones, transcription factors,signaling modules, cytoskeletal proteins and enzymes. Suitable classesof enzymes include, but are not limited to, hydrolases such asproteases, carbohydrases, lipases; isomerases such as racemases,epimerases, tautomerases, or mutases; transferases, kinases,oxidoreductases, and phophatases. Suitable enzymes are listed in theSwiss-Prot enzyme database. Suitable protein backbones include, but arenot limited to, all of those found in the protein data base compiled andserviced by the Research Collaboratory for Structural Bioinformatics(RCSB, formerly the Brookhaven National Lab).

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an antibody refers to an amounteffective in the prevention or treatment of a disorder for the treatmentof which the antibody is effective. A “disorder” is any condition thatwould benefit from treatment with the antibody. This includes chronicand acute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question.

As used herein, the term “variable segment” refers to a portion of anascent peptide which comprises a random, pseudorandom, or definedkernel sequence. A “variable segment” refers to a portion of a nascentpeptide which comprises a random pseudorandom, or defined kernelsequence. A variable segment can comprise both variant and invariantresidue positions, and the degree of residue variation at a variantresidue position may be limited: both options are selected at thediscretion of the practitioner. Typically, variable segments are about 5to 20 amino acid residues in length (e.g., 8 to 10), although variablesegments may be longer and may comprise antibody portions or receptorproteins, such as an antibody fragment, a nucleic acid binding protein,a receptor protein, and the like.

The term “wild-type”, or “wild type”, means that the polynucleotide doesnot comprise any mutations. A “wild type” protein means that the proteinwill be active at a level of activity found in nature and will comprisethe amino acid sequence found in nature.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides a method of integrating therapeutic protein(including antibodies) generation and/or selection, evolution andexpression in a mammalian host for manufacturing in a single system. Inone embodiment of the CIAO method, therapeutic proteins, includingantibodies, are generated, optimized and manufactured in the samemammalian host system. In another embodiment, protein therapeutics arediscovered and manufactured in the same host. This optimizesmanufacturing from the very beginning, saving costs (time andresources).

Historically, discovery of antibodies has been performed in eukaryotic(euk) and prokaryotic (prok) hosts. Typically, in bacteria (E.coli),partial length antibodies are discovered; for example, in phage displaytechnologies, Fabs are recovered and sometimes converted to full lengthdownstream. There are several potential disadvantages to theseapproaches.

In one example, there is some evidence that Fc and Fv regionscommunicate to effect antibody properties, such as binding andexpression. Therefore, when an antibody fragment is optimized for aproperty such as expression, the improvement does not always translateto improved expression in the full length assembled antibody. Forexample, a library of Fc's was created in attempts to find a “holygrail” Fc that could be attached to any Fv to improve expression in anyhost.

In one aspect, codon mutagenesis was performed in the Constant Regionfor optimization of mammalian cell expression. Specifically 326 mutantswere created in the constant region and expressed in HEK 293 and CHO-Scells. Screening was performed by ELISA. Several Fc's met the criteriaof improved expression, and certain optimized Fc's were even identifiedthat transferred positive effects across multiple cell lines; however,when a different Fv was attached to the Fc, the improvement inexpression did not translate. This demonstrates that Fc's and Fv'scommunicate.

In order to avoid unexpected results upon recombination of antibodyfragments, in one preferred aspect, the CIAO method is used to discoverfull length antibody molecules. In another preferred aspect, the CIAOmethod utilizes eukaryotic hosts.

In one embodiment, the eukaryotic system is a mammalian system isselected from one of the group consisting of CHO, HEK293, IM9, DS-1,THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3,HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266,769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2/0cell lines; and mouse splenocytes and rabbit PBMC. In one aspect, themammalian system is selected from a CHO or HEK293 cell line. In onespecific aspect, the mammalian system is a CHO-S cell line. In anotherspecific aspect, the mammalian system is a HEK293 cell line. In anotherembodiment, the eukaryotic system is a yeast cell system. In one aspect,the eukaryotic system is selected from S. cerevisiae yeast cells orpicchia yeast cells.

In another embodiment, the mammalian system is one that that can modifyglycostructure of proteins, such as strains that minimize or eliminatefucose content. Such modifications are believed to enhance antibodybinding to NK cells, and thus modify the ADCC response in potency assaysincreasing the potency of the modified antibodies directed against tumoror infected cells. Several companies offer such technologies to thepublic for manufacturing of modified proteins. For example themammallian system may be one that is lacking in a fucosyltransferaseenzyme such that the cell line produces proteins lacking fucose in theircarbohydrates. For example, the cell lines Ms704, Ms705, and Ms709 lackthe fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), suchthat antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8^(−/−)cell lines were created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioetng 87:614-22). As another example, EP 1,176,195by Hanai et al. describes a cell line with a functionally disrupted FUT8gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the alpha 1,6 bond-related enzyme. Hanai et al. alsodescribe cell lines which naturally have a low enzyme activity foradding fucose to the N-acetylglucosamine that binds to the Fc region ofthe antibody or does not have the enzyme activity, for example the ratmyeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn (297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta (1,4)-N-acetylglucosaminyltransferase III (GnTII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

In another embodiment the system facilitates site-specific, controlledchemical conjugation via modified or unnatural amino acids. Theseinclude host systems which modify natural amino acids to render themamenable to conjugation, or host systems that have been engineered ordesigned to allow chemical conjugation of existing amino acids in theprotein of interest. While a biosynthetic method for a particularunnatural amino acid may not exist in nature, including but not limitedto, in a cell, the invention provides such methods. For example,biosynthetic pathways for unnatural amino acids are optionally generatedin host cell by adding new enzymes or modifying existing host cellpathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. See, for example WO 2002/085923.

Also included in this embodiment are host cells which incorporateunnatural amino acids into proteins, such as strains that containdesigned tRNAs allowing one to direct the incorporation of unnatural,chemically reactive amino acids to specific positions that do notdisrupt desirable characteristics of the protein, such as its ability tobind a receptor.

Chemically aminoacylated suppressor tRNAs can be microinjected orelectroporated into CHO cells and neurons, respectively, and used tosuppress nonsense amber mutations with a series of unnatural amino acids(Monahan et al. (2003), “Site-specific incorporation of unnatural aminoacids into receptors expressed in Mammalian cells,” Chem Biol10:573-580).

Alternatively, heterologous suppressor tRNA/aaRS pairs that do not crossreact with host tRNAs, aaRSs or amino acids (orthogonal tRNA/aaRSs) havebeen engineered to incorporate unnatural amino acids selectively intoproteins. For example, Yokoyama and coworkers modified a Bacillusstearothermophilus amber suppressor tRNA.sub.CUA.sup.Tyr (BstRNA_(CUA)^(Tyr)) and E. coli tyrosyl-tRNA synthetase (EcTyrRS) to incorporate3-iodo-L-tyrosine into proteins in CHO cells (Sakamoto et al. (2002),“Site-specific incorporation of an unnatural amino acid into proteins inmammalian cells,” Nucleic Acids Res 30:4692-4699). Similarly, Zhang andcoworkers engineered an orthogonal Bacillus subtilis suppressortRNA/tryptophanyl-tRNA synthetase pair to incorporate5-hydroxytryptophan into proteins in mammalian cells with high fidelity(Zhang et al. (2004), “Selective incorporation of 5-hydroxytryptophaninto proteins in mammalian cells,” Proc Natl Acad Sci USA101:8882-8887).

Host cells are genetically engineered (including but not limited to,transformed, transduced or transfected) with for example, the codingregions for an orthogonal tRNA, an orthogonal tRNA synthetase, and theprotein to be derivatized; these are operably linked to gene expressioncontrol elements that are functional in the desired host cell.

These methods involve generating a translational machinery thatfunctions independently of the synthetases and tRNAs endogenous to thetranslation system (and are therefore sometimes referred to as“orthogonal”). Typically, the translation system comprises an orthogonaltRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS).Typically, the O-RS preferentially aminoacylates the O-tRNA with atleast one non-naturally occurring amino acid in the translation systemand the O-tRNA recognizes at least one selector codon that is notrecognized by other tRNAs in the system. The translation system thusinserts the non-naturally-encoded amino acid into a protein produced inthe system, in response to an encoded selector codon, thereby“substituting” an amino acid into a position in the encoded polypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. Methods for generating tRNAs and tRNA synthetases which useamino acids that are not encoded in naturally-occurring systems aredescribed in, e.g., U.S. Pat. No. 7,045,337 and U.S. Patent ApplicationPublication U.S. Pat Nos. 7,083,970, and 8,030,028.

Conjugation of stabilizers or toxins to the unnatural amino acids can becontrolled. Various conjugates might be desirable. For example,polyethylene glycol, which allows a slowed clearance of the carriedprotein from the blood is one example. An efficient method for theselective modification of proteins involves the genetic incorporation ofunnatural amino acids, including but not limited to, containing an azideor alkynyl moiety into proteins in response to a selector codon. Theseamino acid side chains can then be modified by, including but notlimited to, a Huisgen [3+2]cycloaddition reaction with, including butnot limited to, alkynyl or azide derivatives, respectively. Because thismethod involves a cycloaddition rather than a nucleophilic substitution,proteins can be modified with extremely high selectivity. Another methodthat can be used is the ligand exchange on a bisarsenic compound with atetracysteine motif, see, e.g., Griffin, et al., (1998) Science281:269-272.

Molecules that can be added to a protein include, but are not limitedto, dyes, fluorophores, crosslinking agents, saccharide derivatives,polymers (including but not limited to, derivatives of polyethyleneglycol), photocrosslinkers, cytotoxic compounds, affinity labels,derivatives of biotin, resins, beads, a second protein or polypeptide(or more), polynucleotide(s) (including but not limited to, DNA, RNA,etc.), metal chelators, cofactors, fatty acids, carbohydrates, and thelike. These molecules can be added to an unnatural amino acid with analkynyl group, e.g, p-propargyloxyphenylalanine, or azido group, e.g.,p-azido-phenylalanine, respectively.

Examples of unnatural amino acids are an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, an unnatural analogue of a tyrosine aminoacid; an unnatural analogue of a glutamine amino acid; an unnaturalanalogue of a phenylalanine amino acid; an unnatural analogue of aserine amino acid; an unnatural analogue of a threonine amino acid; analkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl,alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid,borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone,imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid,or any combination thereof; an amino acid with a photoactivatablecross-linker; a spin-labeled amino acid; a fluorescent amino acid; anamino acid with a novel functional group; an amino acid that covalentlyor noncovalently interacts with another molecule; a metal binding aminoacid; a metal-containing amino acid; a radioactive amino acid; aphotocaged amino acid; a photoisomerizable amino acid; a biotin orbiotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; an amino acidcomprising polyethylene glycol; an amino acid comprising polyether; aheavy atom substituted amino acid; a chemically cleavable orphotocleavable amino acid; an amino acid with an elongated side chain;an amino acid containing a toxic group; a sugar substituted amino acid,e.g., a sugar substituted serine or the like; a carbon-linkedsugar-containing amino acid; a redox-active amino acid; an α-hydroxycontaining acid; an amino thio acid containing amino acid; an α,αdisubstituted amino acid; a β-amino acid; and a cyclic amino acid otherthan proline.

In another embodiment, mammalian cell line creation can be performedcommercially by a contract research or custom manufacturingorganization. For example, for recombinant antibodies or other proteins,Lonza (Lonza Group Ltd, Basel, Switzerland) can create vectors toexpress product using the GS Gene Expression System™ technology witheither CHOK1SV or NSO host cell lines.

In another embodiment, evolution can be performed in prok hosts (such asE.coli) and screens can occur in euk hosts (for example, CHO), as longas screening occurs in same host as manufacturing host.

Selection of Lead Candidate Protein(s)

A variety of methods can be used to discover, generate and/or select oneor more therapeutic protein candidates to be evolved. Molecules selectedcan be existing recombinant proteins, or can come from collections ofrecombinant proteins, including enzymes, hormones and antibodies.Therapeutic proteins can include cloned human enzymes and hormones.Recombinant antibodies can be discovered using any number of generationand screening platforms available. Antibodies can be in any form,including single chain, fully human, Fab, Fv, Fc, bi-functional,chimeric, humanized, or fully human antibodies or fragments thereof. Inone preferred aspect, the CIAO method is used to discover full lengthantibody molecules. Libraries of recombinant antibodies can be generatedand screened using selection or screening systems in optimized ornon-optimized mammalian hosts to yield candidates for evolution. Severaleukaryotic expression systems are published, for example, mammalian oryeast cell systems.

In the method of the present invention, such mammalian expressionsystems, in particular systems using cell surface display of moleculesfor screening and selection, are employed to identify and selectcandidates for manufacturing, or evolution followed by manufacturing.Preferably, such mammalian hosts are Fibroblast cells (3T3, mouse;BHK21, Syrian hamster) Epithelial cells (MDCK, dog; Hela, human; PtK1,rat kangaroo) Plasma cells ((SP2/0 and NS0, mouse) Kidney cells (293,human; COS, monkey) Ovary cells (CHO, Chinese hamster) Embryonic cells(R1 and E14.1, mouse; H1 and H9, human; PER C.6, human). Cell surfacedisplay technology is employed to display proteins on the surface of themammalian cells for screening. Proteins are cloned as fusions withmembrane molecules which when expressed display the proteins on thesurface of the cells for rapid, high-throughput screening, for example.

In certain embodiments, the disclosure provides a method of providing anoptimized antibody. In one embodiment, an antigen is selected and ahuman antibody library is generated and expressed in a mammalian system,for example, CHO-S cells. The library is screened to identify fullyhuman antibody hits, for example, by fluorescence activated cell sorting(FACS). The fully human antibody hits are then furtherscreened/characterized by any relevant assay for at least onepredetermined property, characteristic or activity. In one aspect,evolved molecules are screened for multiple characteristicssimultaneously, for example, improved function and expression. Therelevant assay can comprise for example, ELISA, or an array technology.A template antibody is selected from the human antibody hits. Any methodof evolution is performed on the template antibody, or a fragmentpolypeptide thereof, to prepare a set of mutant antibodies. In oneaspect, the evolution is performed by a method of comprehensive proteinengineering to produce the set of mutant antibodies. The method ofcomprehensive protein engineering can be selected, for example, from oneor a combination of Comprehensive Positional Evolution (CPE™),Comprehensive Protein Synthesis (CPS™), Flex Evolution, SynergyEvolution, Comprehensive Positional Insertion evolution (CPI™), orComprehensive Positional Deletion evolution(CPD™). In another aspect,the mutant antibodies are expressed in the same mammalian system used togenerate the human antibody library.

The set of mutant antibodies are characterized/screened for at least onepredetermined property, characteristic or activity. In one aspect, theset of mutant antibodies is screened simultaneously, for example, forimproved function and expression.

Screening may include screening to eliminate mutant antibodies thatexhibit an undesirable, inappropriate aberrant or abnormal immuneresponse. An undesirable or aberrant immune response, inflammation orinflammatory response is greater or less than desired or physiologicallynormal. An undesirable immune response, inflammation or inflammatoryresponse can be a normal response, function or activity, that isundesired or inappropriate. Thus, normal immune responses, inflammationand inflammatory responses considered undesirable or inappropriate, evenif not aberrant, are included within the meaning of these terms. Anundesirable immune response, inflammation or inflammatory response canalso be an aberrant response, function or activity. An aberrant immuneresponse, inflammation or inflammatory response is abnormal.

Screening conditions can also include conditions that that mimicmanufacturing or formulation conditions. Examples of such conditions areprovided herein in the examples and in the text below relating toformulations.

In one aspect, a molecule specific database in the form of a functionalpositional map (an EvoMap™) is used for additional optimization by oneor more protein evolution techniques known in the art; followed byidentification and characterization of up-mutants. An optimized antibodyis selected by comparison to the template antibody with respect to theat least one predetermined property, characteristic or activity.

In one aspect, gram scale expression of the selected optimized antibodyis performed; followed by non-GLP toxicology. Stable cell linetransfection, process development and generation of a Master Cell Bankis performed. GMP manufacturing is performed in the same mammaliansystem used to generate the human antibody library, for example, CHO-Scells, to result in an optimized therapeutic recombinant mABs.

In another embodiment, the CIAO method starts from selection of atemplate hybridoma or recombinant antibody; evolution of the antibody toprovide a set of mutant antibodies which are screened by use of antibodycell surface display in a mammalian cell system; and manufacturing isperformed in the same mammalian cell system used for screening.

In another embodiment, the selected template hybridoma/recombinantantibody is humanized and screened in the manufacturing host, followedby production in the manufacturing host, wherein the step ofoptimization (evolution) is omitted altogether.

In other aspects of the present invention, downstream expressionoptimization in manufacturing hosts is performed by evolving the Fcregion of the antibody, silent codons in the antibody, and/or the vectorand/or host genes used in protein expression. In one aspect, an Fclibrary is generated by any evolutionary technique. In one specificaspect of expression optimization, CPE is performed on Fc domain of anantibody to create a library of Fc mutants which can be used to selectan optimal partner for any Fv. Optimization is designed for rapidattachment of all Fc CPE variants to each new Fv region. Alternatively,a subset of these Fcs can be used to attach to different Fvs. Each ofthese Fc CPE variant/Fv combinations is screened as a full-lengthantibody expressed in mammalian cells (e.g. CHO, cost-effective media)for optimal expression. Further, CPS can be performed to screen alltheoretical permutations of up to 12 or more of these CPE hits inmammalian cells for expression improvement. Specific desirable codonchanges can also be selected to identify clones with increasedexpression. Silent codons are identified and CPE is performed on thesepositions. This CPE library is screened to identify optimal expressionhits. Further, all theoretical permutations of up to 12 or more CPE hitscan be used in the CPS process to generate a new library that can bescreened in mammalian cells for expression improvement. The top CPSsilent mutation hits are used to customize protein for optimalexpression in a specific cell line and media. This provides opportunityfor biosimilar fine structure control.

Other areas for enhancement of expression include: optimization of thevector, including promoter, splice sites, 5′ and 3′ termini, flankingsequences, reduction of gene deletion and rearrangement, improvement ofhost cell gene activities, optimization of host glycosylating enzymes,and chromosome wide host cell mutagenesis and selection. It has beendemonstrated that 5′ amino acid sequences are important for enhancementof expression.

Evolution of Lead Candidates

Any method of protein evolution can be employed for simultaneousevolution of protein performance and expression optimization.Optimization of protein performance can include improvement of variouscharacteristics such as affinity, pharmacokinetic characteristics,tissue targeting, protein-protein aggregation, addressing high assayvariability and modifying other in vivo characteristics.

Methods for evolving molecules, including selected candidates of thepresent invention, include stochastic and non-stochastic methods.Published methods include random and non-random mutagenesis approaches.Any of these approaches can be employed to evolve properties of thetherapeutic proteins of the present invention toward a desiredcharacteristic, such as better stability in different temperature or pHenvironments, or better expression in a host cell. Other potentiallydesirable properties, such as improved catalytic activity, improvedprotein stability in various conditions, improved selectivity and/orsolubility, and improved expression results by improvement ofcharacteristics such as reduced aggregation can be selected for inevolution experiments.

Evolution is performed directly in a eukaryotic host, such as amammalian cell host or a yeast cell host, that will be used fordownstream production of the therapeutic protein. Candidates can beevolved for optimal expression in the same host used to screen and/orevolve and to manufacture. Expression optimization can be achieved byoptimization of vectors used (vector components, such as promoters,splice sites, 5′ and 3′ termini and flanking sequences), genemodification of host cells to reduce gene deletions and rearrangements,evolution of host cell gene activities by in vivo or in vitro methods ofevolving relevant genes, optimization of host glycosylating enzymes byevolution of relevant genes, and/or by chromosome wide host cellmutagenesis and selection strategies to select for cells with enhancedexpression capabilities. Host cells are further described herein.

Cell surface display expression and screening technology (for example,as defined above) can be employed to screen libraries of evolvedproteins for candidates to be manufactured.

Current methods in widespread use for creating alternative proteins froma starting molecule are oligonucleotide-directed mutagenesistechnologies, error-prone polymerase chain reactions and cassettemutagenesis, in which the specific region to be optimized is replacedwith a synthetically mutagenized oligonucleotide. In these cases, anumber of mutant sites are generated around certain sites in theoriginal sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. Error-prone PCR useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. In a mixture of fragments ofunknown sequence, error-prone PCR can be used to mutagenize the mixture.In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence.

Chimeric genes have been made by joining 2 polynucleotide fragmentsusing compatible sticky ends generated by restriction enzyme(s), whereeach fragment is derived from a separate progenitor (or parental)molecule. Another example is the mutagenesis of a single codon position(i.e. to achieve a codon substitution, addition, or deletion) in aparental polynucleotide to generate a single progeny polynucleotideencoding for a single site-mutagenized polypeptide.

Further, in vivo site specific recombination systems have been utilizedto generate hybrids of genes, as well as random methods of in vivorecombination, and recombination between homologous but truncated geneson a plasmid. Mutagenesis has also been reported by overlappingextension and PCR.

Non-random methods have been used to achieve larger numbers of pointmutations and/or chimerizations, for example comprehensive or exhaustiveapproaches have been used to generate all the molecular species within aparticular grouping of mutations, for attributing functionality tospecific structural groups in a template molecule (e.g. a specificsingle amino acid position or a sequence comprised of two or more aminoacids positions), and for categorizing and comparing specific groupingof mutations. U.S. Pat. No. 7,033,781 entitled “Whole cell engineeringmy mutagenizing a substantial portion of a starting genome, combiningmutations, and optionally repeating” describes a method of evolving anorganism toward desired characteristics. U.S. Pat. No. 6,764,835entitled “Saturation mutagenesis in directed evolution” and U.S. Pat.No. 6,562,594 entitled “Synthetic ligation reassembly in directedevolution” describe methods of exhaustively evolving and screening fordesired characteristics of molecules. Any such methods can be used inthe method of the present invention.

There is a difference between previously known methods of “saturationmutagenesis” and techniques of “comprehensive” evolution preferredherein. Saturation mutagenesis refers to a technique of evolutionwherein every possible change is made at each position of a templatepolynucleotide or template polypeptide; however the change at eachposition is not confirmed by testing, but merely assumed statistically.Comprehensive evolution refers to a technique of evolution wherein everypossible change is made at each position of a template polynucleotide ortemplate polypeptide and the polynucleotide or polypeptide is tested toconfirm the intended change has been made.

Saturation methods are inherently statistical, non-comprehensive methodsand were also never truly comprehensive across all the steps (forexample, across mutant generation, mutant identification, mutant proteinexpression, mutant protein screening, and recombined up-mutantgeneration, identification, expression and screening). In comprehensiveevolution techniques, each molecule is screened and confirmed at boththe first step of mutagenesis, and further at a second step ofrecombining the up-mutants or hits.

Unless the saturation mutagenesis is confirmed by sequencing or someother method, the technique cannot be considered to be comprehensive forseveral possible reasons. For example, 1) cloning systems are not 100%efficient due to due to cloning or synthesis errors, or difficult toclone molecules or 2) some proteins are toxic when expressed and thuscannot be efficiently expressed. Therefore, it is important to confirmby sequencing, or some other technique, at each step. It is useful toscore every step in order to screen for expression, so non-expressingclones don't get designated as “negative” as in previous work, they justget scored non-expressible. Comprehensive techniques are thereforeconsidered to be more pure non-stochastic system than saturationtechniques, as confirmed by the “confirmation” step.

Comprehensive Positional Evolution

Referring to FIG. 1, using a linear peptide as a simple example, in afirst step, a set of naturally occurring amino acid variants (or asubset thereof, or amino acid derivatives) for each codon from position1 to n (n corresponding to the number of residues in the polypeptidechain) is generated by a process referred to herein as ComprehensivePositional Evolution (CPE™). This procedure is repeated for eachpolypeptide chain of the target molecule. A minimum set of amino acidmutations contains only one codon for each of the 19 natural aminoacids. However, it is recognized that each expression system may sufferfrom codon bias, in which insufficient tRNA pools can lead totranslation stalling, premature translation termination, translationframeshifting and amino acid misincorporation. Therefore, for expressionoptimization each set contains up to 63 different codons, including stopcodons. In the next step, the mutations are confirmed by sequencing eachnew molecule. Other methods of confirmation can also be employed.

Each amino acid set is then screened for at least one of:

Improved function

Neutral mutations

Inhibitory mutations

Expression

Compatibility of the clone with the host system.

Preferably, multiple characteristics are screened for simultaneouslysuch as, for example, improved function and expression.

The data for each set are combined for the entire polypeptide chain(s)and a detailed functional map (referred to herein as an EvoMap™) of thetarget molecule is generated. This map contains detailed information howeach mutation affects the performance/expression and/or cloningcapability of the target molecule. It allows for the identification ofall sites where no changes can be made without a loss in proteinfunction (e.g., antigen/receptor binding in case of antibodies). It alsoshows where changes can be made without affecting function. It furtheridentifies changes that result in molecules that do not express in thehost system, and therefore do not assess the effect of the mutation.

A schematic of a hypothetical EvoMap™ is shown in FIG. 1. Each positionon the template is identified as a restricted site (non-mutable), afully mutable site, a partially mutable site or an up-mutant for aspecific amino acid substitution. Each partially mutable site may befurther designated as amenable to substitution with, for example, acharged residue, or a non-polar residue substitution, and anon-expressing clone and/or molecule that cannot be cloned in the hostsystem.

It is possible to utilize the EvoMap™ in order to recognize andrecombine beneficial single amino acid substitutions, and screen tofurther optimize the desired characteristics in the target molecule.However, evolution of certain characteristics may require two or moresimultaneous mutations to become observable. The EvoMap™ may beexploited to efficiently, and cost effectively, produce a set ofmulti-site mutant polypeptides in a non-random fashion. The set ofmulti-site mutant polypeptides can then be screened for multi-siteupmutants.

CPE enables the complete in vivo confirmed protein mutation map.Identification of the entire set of up-mutants enables furthercombinatorial evolution step(s). CPE can be utilized in order to reducethe immunogenicity risk of evolved proteins by the selection ofnon-surface mutations; elimination of T-cell epitopes; and mimicry ofsomatic mutations.

In one aspect, CPE can be used to generate a library of up to 5, 10 or15 amino acids, or up to all 19 amino acids. Changes are made at eachposition in the protein and screened for a desirable characteristic,such as binding affinity or expression, and the Evomap™ is created.Later rounds of mutation and screening can be used to generate the datafor all 19 amino acids. From the map, fully mutable sites areidentified. These sites are useful to identify positions that can bemodified to create a new collection of molecules that can be made andtested for new characteristics. For example, informatics can be employedto identify HLA haplotypes in the sequence, and desired changes can bemade to avoid these haplotypes by making specific targeted changes at“neutral” (“fully mutable”) sites identified from the map, where theprimary characteristic will not be affected. This could potentiallyreduce immunogenicity risk (one could select non-surface mutations,eliminate t-cell epitopes, mimic hypersomatic mutations). Further, themap can show sites for site specific modifications (glycosylation andchemical conjugation) to improve various characteristics. Also,optimization of silent mutations can improve protein expression in avariety of hosts.

Synergy Evolution

In one embodiment of the present invention, an EvoMap™ is generated andutilized for Synergy Evolution, as shown in FIG. 2. In SynergyEvolution, simultaneous mutation at 2 -20 selected sites may be combinedto produce a combinatorial effect. The EvoMap™ of the templatepolypeptide is used to select specific single amino acid point mutationsfor assembly to multi-site polypeptide mutations.

In Synergy Evolution, non-deactivating amino acid point mutations areselected from within partially mutable sites that are near non-mutablesites on the EvoMap™. In one aspect, the selected non-deactivating pointmutations are adjacent to non-mutable sites. In Synergy Evolution,simultaneous mutation of amino acids at two to 20 of the selected sitesis performed for combinatorial effects. In one aspect, recombination oftwo to 20 selected mutations is used to produce a codon variant librarycoding for a population of multi-site mutant polypeptides. Followingcloning and expression, the multi-site mutant polypeptides produced arethen screened for at least one predetermined property, characteristic oractivity compared to the template polypeptide. In this manner,multi-site upmutant polypeptides can be identified. In one aspect,multi-site mutant polypeptides are produced by combinatorial proteinsynthesis. One advantage of Synergy Evolution is that it does notrequire a protein x-ray crystal structure to direct evolution of thetemplate polypeptide. This technique is useful particularly for proteinswith high assay variation and other multi-site effects.

According to the present invention, applications of Synergy Evolutioninclude, but are not limited to evolution of complex molecularmechanistic changes, evolution of proteins with high assay variation,evolution of protein specificity, improvement of expression in variousexpression hosts, improvement of protein catalytic activity, stability,and pH optimization. Synergy Evolution is applicable to all proteintherapeutic types including, but not limited to, hormones, enzymes,cytokines and antibodies.

In one aspect of the present invention, Synergy Evolution can be used tooptimize one or more aspects of a polypeptide which is a portion of aprotein molecule. The protein molecule can be assembled by ligating oneor more mutant nucleic acids coding for polypeptides with zero, one ormore nucleic acids coding for framework polypeptides to create a variantprotein by cloning, translation and expression techniques known in theart. In one aspect, a framework polypeptide is derived from a wild-typeprotein molecule. In this aspect, Synergy Evolution can be used inconjunction with antibody humanization techniques. For example, a mousemonoclonal antibody may be selected for evolution and humanization. TheCDR regions of the antibody are cloned and sequenced and individual CDRregions (CDR1, CDR2, CDR3) may be synthesized and ligated to othernucleotides coding for human antibody framework polypeptides, followedby production of a human variant IgG library. The human variant IgGlibrary is then screened for at least one property, for example two ormore properties including improved function and expression, compared tothe mouse mAb. In another aspect, a framework polypeptide is anartificial scaffold polypeptide. Specific techniques of ds DNA fragmentpreparation, ligation and assembly of nucleic acids, cloning,transfection, expression, solid phase synthesis of libraries, solutionphase synthesis of libraries, comprehensive positional evolution,combinatorial protein synthesis, quantification of expression by ELISAquantification and ™-galactosidase assay, and functional ELISA arepresented in the examples section.

In another embodiment of the invention, Synergy Evolution can be used toenhance binding affinity of an antibody. In this embodiment,optimization of the antibody variable region may be performed. Forexample, for the production of antibody mutants, CPE is performed forlight chain and heavy chain variable regions of a selected antibody andan EvoMap™ is generated. Mutants are selected for reassembly; forexample, variants of the light chain are selected and variants of theheavy chain are selected for assembly. Non-deactivating amino acid pointmutations are selected from within partially mutable sites that are nearnon-mutable sites. The reassembly technology utilizing CPS can be usedto create a library of heavy chains. The light chain variants can becombined with the heavy chain variants, cloned, expressed and thevariants are screened as full IgGs from eukaryotic cell linesupernatants. Binding affinity for certain variants is assessed by, forexample, use of ELISA, BlAcore and/or Sapidyne instrumentation assays,or other techniques known to one in the art.

Flex Evolution

In another embodiment, the CPE/EvoMap may be used to identify andexploit fully mutable sites. In one aspect, exploitation of multiplefully mutable sites is termed Flex Evolution and is used to maketargeted changes such as introduction of sites for glycosylation (e.g.codons for amino acids for N- or O-linked glycosylation; Asn withinconsensus sequence Asn-Aa-Ser-Thr or Ser/Thr) and chemical conjugation.Flex evolution may also be used in design of protease cleavage sites,introduction of tags for purification and/or detection, site-specificlabeling, and the like. Further, codon optimization of silent mutationsmay be utilized for improvement of protein expression. In thisembodiment, termed Flex Evolution, following protein expression, themutant polypeptide libraries produced are rescreened for at least onepredetermined property, characteristic or activity compared to thetemplate polypeptide. In one aspect, the predetermined property includesreduction of protein-protein aggregation, enhancement of proteinstability, or increased protein solubility. In one aspect, the mutantpolypeptide libraries are screened for two or more propertiessimultaneously. In another aspect, any eukaryotic expression systemwhich glycosylates may be used for the introduction of glycosylationsites, such as, for example, mammalian, plant, yeast, and insect celllines.

In Flex Evolution, evaluation of bioinformatics and protein x-raycrystal structures of related proteins, or the template protein orpolypeptide, is useful for template optimization. In one aspect,selected sites are not at contact residues. In another aspect, selectionof non-surface protein mutations allows for reduced immunogenicity risk.

Applications of Flex Evolution include, bit are not limited to,reduction of protein-protein aggregation, improvement of proteinsolubility, optimization of pharmacokinetics via glycosylationlibraries, optimization of protein secondary and tertiary structure anddeimmunization of antigenic sites directly via either mutation sets orindirectly through glycosylation masking.

In one aspect of Flex Evolution, an EvoMap™ is utilized to identifyfully mutable sites, CPS generation is performed with insertion ofglycosylating residues to fully mutable sites (or silent mutations fortranslation effects), and screening of combinatorial glycosylatedlibrary is performed by analytical analysis (e.g. Mass Spectroscopyanalysis, Dynamic Light Scattering), immunogenicity reduction (bybioinformatics or assay), and/or pharmacokinetic analysis (e.g. inFoxnlnu mice).

In one aspect, Flex evolution may be used for deimmunization toeliminate immunogenicity while maintaining function. Flex Evolutiondeimmunization can be performed by masking immunogenicity withglycosylation, identifying human hypersomatic mutation spectra aminoacid substitutions that may eliminate immunogenicity while maintainingfunction, reduction of dose for evading immunogenicity potential, andminimization of non-surface amino acid residue changes. Further,immunogenicity databases and algorithms can be used to identify andreplace potential MHC binding epitopes. In one aspect, in silicomodification prediction is coupled with CPE/CPS data to generatevariants.

Reduced propensity to generate T-cell epitopes and/or deimmunization maybe measured by techniques known in the art. Preferably, deimmunizationof proteins may be tested in vitro by T cell proliferation assay. Inthis assay PBMCs from donors representing >80% of HLA-DR alleles in theworld are screened for proliferation in response to either wild type ordeimmunized peptides. Ideally cell proliferation is only detected uponloading of the antigen-presenting cells with wild type peptides.Additional assays for deimmunization include human in vitro PBMCre-stimulation assays (e.g. interferon gamma (TH1) or IL4 (TH2) ELISA.Alternatively, one may test deimmunization by expressing HLA-DRtetramers representing all haplotypes. In order to test if de-immunizedpeptides are presented on HLA-DR haplotypes, binding of e.g.fluorescence-labeled peptides on PBMCs can be measured. Measurement ofHLA Class I and Class II transgenic mice for responses to target antigen(e.g. interferon gamma or IL4). Alternatively epitope library screeningwith educated T cells (MHCI 9mer; MHCII 20mer) from PBMC and/ortransgenic mouse assays. Furthermore, deimmunization can be proven bydetermining whether antibodies against the deimmunized molecules havebeen generated after administration in patients.

In another embodiment, the Flex Evolution techniques of the presentinvention can be utilized for expression optimization. In one aspect,the present invention discloses the utilization of protein engineeringmethods to develop silent mutation codon optimized Fc variants withimproved expression in eukaryotic cells. A silent mutation is one inwhich the variation of the DNA sequence does not result in a change inthe amino acid sequence of the protein. In one aspect, codon mutagenesisis performed in the constant region for optimization of eukaryotic cellexpression. A codon optimized Fc variant with improved expressionproperties while retaining the capacity to mediate effector functionsimproves the production of therapeutic antibodies. In this aspect, forexample, a constant region of an antibody molecule can be evolved forscreening in different expression hosts, for example, mammalian celllines expression screening utilizing CHO, HEK293 and COS-7. One exampleof expression optimization by codon mutagenesis in the constant regionfor mammalian cell expression is shown in FIG. 3 and described inExample 19. The expression levels shown are each an average of 4 datapoints, and confirmed over multiple experiments. Multiple cell linecapability was demonstrated for first mutant tested in HEK293 and CHOcell line expression systems.

In addition, the EvoMap™ may be used to generate 3-dimensionalcomputational molecular models of the oligopeptide, or specific regionsthereof, to explore the structural mechanisms involved in, e.g.,antibody-epitope specificity and stability. A hypotheticalthree-dimensional EvoMap™ is shown in FIG. 13.

The information in EvoMap can also be combined with structuralinformation (if available) to select e.g., only surface residues formutations to increase solubility/decrease aggregation.

Comprehensive Positional Insertion Evolution

In one embodiment, the disclosure provides methods of identifying andmapping mutant polypeptides formed from, or based upon, a templatepolypeptide. Referring to FIG. 8, using a linear peptide as a simpleexample, in a first step, a set of naturally occurring amino acidvariants (or a subset thereof, or amino acid derivatives) for each codonfrom position 2 to n (n corresponding to the number of residues in thepolypeptide chain) is generated by a process referred to herein asComprehensive Positional Insertion (CPI™) evolution.

In CPI™, an amino acid is inserted after each amino acid throughout atemplate polypeptide one at a time to generate a set of lengthenedpolypeptides. CPI can be used to insert 1, 2, 3, 4, or up to 5 new sitesat a time. Each of the 20 amino acids is added at each new position, oneat a time, creating a set of 20 different molecules at each new positionadded in the template. In this case, position 1, which is methionine andinvariant, is skipped. This procedure is repeated for each polypeptidechain of the target molecule. A minimum set of amino acid mutationscontains only one codon for each of the 20 natural amino acids.

The present invention relates to methods of identifying and mappingmutant polypeptides formed from, or based upon, a template polypeptide.Typically, the polypeptide will comprise n amino acid residues, whereinthe method comprises (a) generating n+[20×(n-1)] separate polypeptides,wherein each polypeptide differs from the template polypeptide in thatit has inserted after each position in the template each of the 20 aminoacids one at a time (as illustrated in FIG. 1); assaying eachpolypeptide for at least one predetermined property, characteristic oractivity; and (b) for each member identifying any change in saidproperty, characteristic or activity relative to the templatepolypeptide.

In one embodiment, one or more regions are selected for mutagenesis toadd one position at a time as described above. In such case, nrepresents a subset or region of the template polypeptide. For example,where the polypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto mutagenesis to add one position at a time in the template polypeptideafter each position.

The invention thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating n+[20×(n-1)] separate antibodies, wherein each antibodydiffers from the template antibody in that has inserted a singlepredetermined position, one at a time, after each position in thetemplate sequence; (b) assaying each set for at least one predeterminedproperty, characteristic or activity; and (c) for each memberidentifying any change in a property, characteristic or activityrelative to the template polypeptide. For antibodies, the predeterminedproperty, characteristic or property may be binding affinity and/orimmunogenicity, for example.

In addition, provided are methods of producing a set of mutantantibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n+[20×(n-1)]separate antibodies, wherein each antibody differs from the templateantibody in that it has an extra amino acid added at a singlepredetermined position of the CDR. In another embodiment, the antibodycomprises six CDRs, and together the CDRs comprise n amino acidresidues.

In another embodiment, the new lengthened polypeptides described aboveare further mutated and mapped after screening to identify a change in aproperty, characteristic or activity relative to the shortenedpolypeptide. Typically, the lengthened polypeptide will comprise n aminoacid residues, wherein the method comprises (a) generating n (n-1 in thecase where the initial residue is methionine) separate sets ofpolypeptides, each set comprising member polypeptides having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the polypeptide; wherein each set of polypeptides differs inthe single predetermined position; assaying each set for at least onepredetermined property, characteristic or activity; (b) for each memberidentifying any change in said property, characteristic or activityrelative to the template polypeptide; and optionally (c) creating afunctional map reflecting such changes. Preferably, the number ofdifferent member polypeptides generated is equivalent to n×X (or[n-1]×X, as the case may be).

In the alternative, the method comprises generating a single populationcomprising the sets of mutated polypeptides from the lengthenedpolypeptides. In this embodiment, the entire new population is screened,the individual members identified, and the functional map generated.

Typically, where each naturally occurring amino acid is used, X will be19 (representing the 20 naturally occurring amino acid residues andexcluding the particular residue present in a given position of thetemplate polypeptide). However, any subset of amino acids may be usedthroughout, and each set of polypeptides may be substituted with all ora subset of the total X used for the entire population.

However, it is recognized that each expression system may suffer fromcodon bias, in which insufficient tRNA pools can lead to translationstalling, premature translation termination, translation frameshiftingand amino acid misincorporation. Therefore, for expression optimizationeach set contains up to 63 different codons.

Each amino acid set is then screened for at least one, and preferablytwo or more, desirable characteristic such as improved function; neutralmutations, inhibitory mutations, and expression.

In one aspect, the lengthened polypeptides can be mapped to identify achange in a property, characteristic or activity resulting in theshortened polypeptides relative to the “wildtype”. The data for each setare combined for the entire polypeptide, or “target molecule”. Hits fromthe screening of the lengthened polypeptides (target molecules) can thenbe used for further comprehensive mutagenesis chain(s) and screening asdescribed herein. The data from mutagenesis provides a detailedfunctional map (referred to herein as an EvoMap™) of the target moleculeis generated. This map contains detailed information how each mutationaffects the performance/expression of the target molecule. It allows forthe identification of all sites where no changes can be made without aloss in protein function (or antigen/receptor binding in case ofantibodies). It also shows where changes can be made without affectingfunction.

In another aspect, CPE can be used to generate a library of 5, 10, up to15, or up to all 19 amino acids at each position of interest.

Comprehensive Positional Deletion Evolution

Comprehensive Positional Deletion Evolution (CPDTM) relates to methodsof identifying and mapping mutant polypeptides formed from, or basedupon, a template polypeptide. CPD evolution deletes every amino acidthrough the protein one position at a time. Typically, the polypeptidewill comprise n amino acid residues, wherein the method comprises (a)generating n-1 (n-2 in the case where the initial residue is methionine)separate polypeptides, wherein each polypeptide differs from thetemplate polypeptide in that it lacks a single predetermined position;assaying each polypeptide for at least one predetermined property,characteristic or activity; and (b) for each member identifying anychange in said property, characteristic or activity relative to thetemplate polypeptide.

In one embodiment of CPD evolution, one or more regions are selected formutagenesis to remove one position at a time. In such case, n representsa subset or region of the template polypeptide. For example, where thepolypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto mutagenesis to remove one position at a time in the templatepolypeptide.

In one embodiment, CPD thus includes methods of mapping a set of mutantantibodies formed from a template antibody having at least one, andpreferably six, complementarity determining regions (CDRs), the CDRstogether comprising n amino acid residues, the method comprising (a)generating (n-1) separate antibodies, wherein each antibody differs fromthe template antibody in that lacks a single predetermined position; (b)assaying each set for at least one predetermined property,characteristic or activity; and (c) for each member identifying anychange in a property, characteristic or activity relative to thetemplate polypeptide. For antibodies, the predetermined property,characteristic or property may be binding affinity and/orimmunogenicity, for example.

One aspect of CPD evolution includes methods of producing a set ofmutant antibodies formed from a template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n-1 separateantibodies, wherein each antibody differs from the template antibody inthat lacks a single predetermined position of the CDR. In anotherembodiment, the antibody comprises six CDRs, and together the CDRscomprise n amino acid residues.

In another embodiment of CPD evolution, the new shortened polypeptidesdescribed above are further mutated and mapped after screening toidentify a change in a property, characteristic or activity relative tothe shortened polypeptide. Typically, the shortened polypeptide willcomprise n amino acid residues, wherein the method comprises (a)generating n (n-1 in the case where the initial residue is methionine)separate sets of polypeptides, each set comprising member polypeptideshaving X number of different predetermined amino acid residues at asingle predetermined position of the polypeptide; wherein each set ofpolypeptides differs in the single predetermined position; assaying eachset for at least one predetermined property, characteristic or activity;(b) for each member identifying any change in said property,characteristic or activity relative to the template polypeptide; and (c)creating a functional map reflecting such changes. Preferably, thenumber of different member polypeptides generated is equivalent to n×X(or [n-1]×X, as the case may be).

In the alternative, the CPD method comprises generating a singlepopulation comprising the sets of mutated polypeptides from theshortened polypeptides. In this embodiment, the entire new population isscreened, the individual members identified, and the functional mapgenerated. Typically, where each naturally occurring amino acid is used,X will be 19 (representing the 20 naturally occurring amino acidresidues and excluding the particular residue present in a givenposition of the template polypeptide). However, any subset of aminoacids may be used throughout, and each set of polypeptides may besubstituted with all or a subset of the total X used for the entirepopulation.

Any mutational or synthetic means may be used to generate the set ofmutants in CPD evolution. In one embodiment, the generation ofpolypeptides comprises (i) subjecting a codon-containing polynucleotideencoding for the template polypeptide to polymerase-based amplificationusing a 64-fold degenerate oligonucleotide for each codon to bemutagenized, wherein each of the 64-fold degenerate oligonucleotides iscomprised of a first homologous sequence and a degenerate N,N,N tripletsequence, so as to generate a set of progeny polynucleotides; and (ii)subjecting the set of progeny polynucleotides to clonal amplificationsuch that polypeptides encoded by the progeny polynucleotides areexpressed.

In one embodiment of CPD evolution, the entire shortened polypeptide issubjected to saturation mutagenesis. In another embodiment, one or moreregions are selected for saturation mutagenesis. In such case, nrepresents a subset or region of the template polypeptide. For example,where the polypeptide is an antibody, the entire antibody or one or morecomplementarity determining regions (CDRs) of the antibody are subjectedto saturation mutagenesis.

The CPD evolution disclosure thus includes methods of mapping a set ofmutant antibodies formed from a shortened template antibody having atleast one, and preferably six, complementarity determining regions(CDRs), the CDRs together comprising n amino acid residues, the methodcomprising (a) generating n separate sets of antibodies, each setcomprising member antibodies having X number of different predeterminedamino acid residues at a single predetermined position of the CDR;wherein each set of antibodies differs in the single predeterminedposition; and the number of different member antibodies generated isequivalent to n×X; (b) assaying each set for at least one predeterminedproperty, characteristic or activity; (c) for each member identifyingany change in a property, characteristic or activity relative to thetemplate polypeptide; and (d) creating a structural positional map ofsuch changes. For antibodies, the predetermined property, characteristicor property may be binding affinity and/or immunogenicity. As set forthabove, in the alternative a single population comprising all sets ofmutated antibodies may be generated.

In addition, provided are methods of producing a set of mutantantibodies formed from a shortened template antibody having at least onecomplementarity determining region (CDR), the CDR comprising n aminoacid residues, the method comprising: (a) generating n separate sets ofantibodies, each set comprising member antibodies having X number ofdifferent predetermined amino acid residues at a single predeterminedposition of the CDR; wherein each set of antibodies differs in thesingle predetermined position; and the number of different memberantibodies generated is equivalent to n×X. In another embodiment,antibody comprises six CDRs, and together the CDRs comprise n amino acidresidues.The CPD™ evolution method includes a functional positional map(EvoMap™) made by the methods described herein. In an additionalembodiment, certain residues particularly sensitive to change may be soindicated on the EvoMap™. Further optimization may be implemented bymaking additional mutational changes at positions outside of thesesensitive positions. It is also possible to utilize the EvoMap™ in orderto recognize and recombine beneficial single amino acid substitutions,and screen to further optimize the desired characteristics in the targetmolecule, in a process called Combinatorial Protein Synthesis (CPS™)

Combinatorial Protein Synthesis

Combinatorial Protein Synthesis (CPS™) involves combining individualhits from CPE, CPI, CPD, or any other evolutionary technique to combinetwo or more mutations. CPS is used to synthesize proteins with combinedmutations which are then screened for optimized gene and proteincharacteristics. A schematic of CPS™ is shown in FIG. 3. In one aspect,two or more point mutations which result in up-mutants or neutralmutations are combined in CPS.

In one embodiment CPE is combined with CPS to create mutants, which arescreened for the desired property. In one aspect, time and resources canbe saved in the CPE process by changing 2 aa or 3 aa or 4 aas at a timeversus one at a time; so if the number of aa's in the protein is N, thetotal number generated and screened for 2 aa at a time would be(20²)×½N; 3 at a time would be (20³)×⅓N, etc. For example, in onespecific aspect, (in the 2aa example): 1^(st) aa at 1^(st) aa positionis combined with all 20 at the 2^(nd) aa position and all the other aa'sremain the same, then the 2^(nd) aa at 1^(st) aa position is combinedwith all 20 at the 2^(nd) aa position and all other aa's remain thesame. The entire population is screened for up mutants and then mutationat the second set of the next two aa's down the line is performed. In asimilar aspect, this can be performed for 3aas at a time or 4aas at atime. In another aspect, optionally follow the CPE process with CPS ofupmutants (including any subset thereof).

In one aspect, non-natural amino acids can be incorporated into theprocess (so all 19 other amino acids, or a subset thereof, plusnon-natural amino acids) by using novel technologies such as thequadruplet codon described in the attached and related papers. Neumannet al. Encoding multiple unnatural amino acids via evolution of aquadruplet-decoding ribosome Nature 464, 441-444 (14 Feb. 2010). In thisaspect CPE/CPS is performed for incorporation of non-natural aminoacids.

In a further aspect, the entire CPE library is created synthetically(synthesizing all the molecules on commercially available machines). Inthe event the synthesis machine cannot create large enough strands,fragments are synthesized and then ligated to generate full lengthmolecules. This library is screened and followed with CPS to combinedesired mutations. This is a two step process wherein CPE is followed byCPS, not one step of only CPE.

In another aspect, a CPE library is generated and screened, thenfollowed by CPS combining up mutants as follows: if there are 10up-mutants, test a single molecule with all 10 changes, then test allversions of 9 mutations, then 8, 7, 6, 5 etc. until one of the groupsdoes not find an improved molecule over any in the previous group. Oncean improved molecule is identified the process can be terminated.

In a further aspect, CPE is performed to identify up-mutants and neutralmutations for affinity and expression, then CPS is performed withcombinations of up mutants and neutral mutations, and the library isrescreened for further improvements in characteristics such as function,affinity and/or expression.

In a further aspect, CPE is performed on codons of the Fc or otherdomain for glycosylation changes.

In another aspect, CPE or CPE combined with CPS of microRNA's or intronscan be performed.

In a further aspect, CPE or CPE combined with CPS of rodent antibodyCDRs is performed, then screened for up-mutants, followed byhumanization.

In one aspect, CPE or CPE combined with CPS is performed to producealternative intermediate nucleotides that lead to the desired mutationin the final reaction, for example, a methylated cytosine that convertsto a uracil.

In one aspect, CPE or CPE combined with CPS plus informatics is utilizedfor converting mouse CDR's to human CDR's and vice versa.

In one aspect, CPE or CPE combined with CPS is utilized with 2 and 3mutations spaced throughout the protein.

In another aspect, CPE or CPE combined with CPS are used in a dual chainvector for screening evaluation for increased sensitivity.

In a further aspect, CPE or CPE combined with CPS is utilized forselecting for allosteric changes.

Any of several screening techniques can be used to evaluate CPE/CPSmutants. In one aspect CPE or CPE combined with CPS mutants can besecreted and displayed in eukaryotic hosts. Alternatively, CPE or CPEcombined with CPS mutants can be produced in E.coli and screened ineukaryotic hosts. In another aspect, CPE is performed starting at 15aaor 10aa and followed by CPS; then followed up with the rest of theremaining 19aa. In another aspect, CPE or CPE combined with CPS isutilized for evolving proteins specifically with non-surface amino acidchanges. In one aspect, CPE for can be utilized for multi-dimensionalepitope mapping. In another aspect, CPE or CPE combined with CPSscreening can be performed transiently in eukaryotic cells. In a furtheraspect, CPE or CPE combined with CPS is performed, then sequencing andarray of all clones is performed, for example, in a chip based orwell-based format for expression and screening. In another aspect, CPEor CPE combined with CPS is utilized for evolving metal ion coordinationby selecting in varying ion concentrations. In a further aspect, CPE orCPE combined with CPS is performed, and proteins are expressed andscreened in cell free conditions and in non-human living organisms. Inone aspect, CPE or CPE combined with CPS screening of stem cells isperformed for varying effects on differentiation and protein and RNA andmRNA expression. In a further aspect, CPE or CPE combined with CPSmultiplex screening is performed for multiple protein characteristicslike expression and binding. In another aspect, CPE or CPE combined withCPS is performed on template molecules involved in brain transport andmembrane crossing; and mutants are screened for improvedcharacteristics. In one aspect, CPE or CPE combined with CPS mutants arescreened for protein hygroscopic characteristics. In another aspect, CPEor CPE combined with CPS mutants are assayed for selecting dynamicproteins. In one aspect, CPE or CPE combined with CPS screening isperformed outside of the target condition to identify mutants withintarget condition and vice versa.

In one embodiment, any of the above aspects of CPE or CPE combined withCPS are utilized in combination with a method selected from CPI, CPD,and CPD with CPI combination.

In another embodiment, any of the above aspects of CPE or CPE combinedwith CPS are utilized in combination with a method selected from Flexevolution and Synergy evolution performed from a template.

The term “template” may refer to a base polypeptide or a polynucleotideencoding such polypeptide. As would be appreciated by one of skill inthis art, any template may be used in the methods and compositions ofthe present invention. Templates which can be mutated and therebyevolved can be used to guide the synthesis of another polypeptide orlibrary of polypeptides as described in the present invention. Asdescribed in more detail herein, the evolvable template encodes thesynthesis of a polypeptide and can be used later to decode the synthetichistory of the polypeptide, to indirectly amplify the polypeptide,and/or to evolve (i.e., diversify, select, and amplify) the polypeptide.The evolvable template is, in certain embodiments, a nucleic acid. Incertain embodiment of the present invention, the template is based on anucleic acid. In other embodiments, the template is a polypeptide.

The nucleic acid templates used in the present invention are made ofDNA, RNA, a hybrid of DNA and RNA, or a derivative of DNA and RNA, andmay be single- or double-stranded. The sequence of the template is usedto encode the synthesis of a polypeptide, preferably a compound that isnot, or does not resemble, a nucleic acid or nucleic acid analog (e.g.,an unnatural polymer or a small molecule). In the case of certainunnatural polymers, the nucleic acid template is used to align themonomer units in the sequence they will appear in the polymer and tobring them in close proximity with adjacent monomer units along thetemplate so that they will react and become joined by a covalent bond.In certain other embodiments, the template can be utilized to generatenon-natural polymers by PCR amplification of a synthetic DNA templatelibrary consisting of a random region of nucleotides.

It will be appreciated that the template can vary greatly in the numberof bases. For example, in certain embodiments, the template may be 10 to10,000 bases long, preferably between 10 and 1,000 bases long. Thelength of the template will of course depend on the length of thecodons, complexity of the library, length of the unnatural polymer to besynthesized, complexity of the small molecule to be synthesized, use ofspace sequences, etc. The nucleic acid sequence may be prepared usingany method known in the art to prepare nucleic acid sequences. Thesemethods include both in vivo and in vitro methods including PCR, plasmidpreparation, endonuclease digestion, solid phase synthesis, in vitrotranscription, strand separation, etc. In certain embodiments, thenucleic acid template is synthesized using an automated DNA synthesizer.

As discussed above, in certain embodiments of the invention, the methodis used to synthesize polypeptides that are not, or do not resemble,nucleic acids or nucleic acid analogs. Thus, in certain embodiments ofthe present invention, the nucleic acid template comprises sequences ofbases that encode the synthesis of an unnatural polymer or smallmolecule. The message encoded in the nucleic acid template preferablybegins with a specific codon that bring into place a chemically reactivesite from which the polymerization can take place, or in the case ofsynthesizing a small molecule the “start” codon may encode for ananti-codon associated with a small molecule scaffold or a firstreactant. The “start” codon of the present invention is analogous to the“start” codon, ATG, which encodes for the amino acid methionine.

In yet other embodiments of the invention, the nucleic acid templateitself may be modified to include an initiation site for polymersynthesis (e.g., a nucleophile) or a small molecule scaffold. In certainembodiments, the nucleic acid template includes a hairpin loop on one ofits ends terminating in a reactive group used to initiate polymerizationof the monomer units. For example, a DNA template may comprise a hairpinloop terminating in a 5′-amino group, which may be protected or not.From the amino group polymerization of the unnatural polymer maycommence. The reactive amino group can also be used to link a smallmolecule scaffold onto the nucleic acid template in order to synthesizea small molecule library.

To terminate the synthesis of the unnatural polymer a “stop” codonshould be included in the nucleic acid template preferably at the end ofthe encoding sequence. The “stop” codon of the present invention isanalogous to the “stop” codons (i.e., TAA, TAG, TGA) found in mRNAtranscripts. These codons lead to the termination of protein synthesis.In certain embodiments, a “stop” codon is chosen that is compatible withthe artificial genetic code used to encode the unnatural polymer. Forexample, the “stop” codon should not conflict with any other codons usedto encode the synthesis, and it should be of the same general format asthe other codons used in the template. The “stop” codon may encode for amonomer unit that terminates polymerization by not providing a reactivegroup for further attachment. For example, a stop monomer unit maycontain a blocked reactive group such as an acetamide rather than aprimary amine In yet other embodiments, the stop monomer unit comprisesa biotinylated terminus providing a convenient way of terminating thepolymerization step and purifying the resulting polymer.

In one embodiment, mutagenized DNA products are used directly as thetemplate for in vitro synthesis of the corresponding mutant proteins.Because of the high efficiency with which all 19 amino acidsubstitutions can be generated at a single residue, it is possible toperform saturation mutagenesis on numerous residues of interest, eitherindependently or in combination with other mutations within the protein.As used herein, “complete saturation” mutagenesis is defined asreplacing a given amino acid within a protein, with the other 19naturally-occurring amino acids. For example, gene site saturationmutagenesis, which systematically explores minimally all possible singleamino acid substitutions along a protein sequence, is disclosed in Kretzet al., Methods in Enzymology, 2004, 388:3-11; Short, U.S. Pat. No.6,171,820; and Short, U.S. Pat. No. 6,562,594, each of which isincorporated herein by reference.

In one aspect, this invention provides for the use of codon primers(containing a degenerate N,N,G/T sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (see U.S. Pat. No. 6,171,820; see also, U.S.Pat. No. 5,677,149, each incorporated herein by reference). The oligosused are comprised contiguously of a first homologous sequence, adegenerate N,N,G/T sequence, and preferably but not necessarily a secondhomologous sequence. The downstream progeny translational products fromthe use of such oligos include all possible amino acid changes at eachamino acid site along the polypeptide, because the degeneracy of theN,N,G/T sequence includes codons for all 20 amino acids.

Codon usage is one of the important factors in eukaryotic geneexpression. The frequencies with which different codons are used varysignificantly between different hosts, and between proteins expressed athigh or low levels within the same organism. The most likely reason forthis variation is that preferred codons correlate with the abundance ofcognate tRNAs available within the cell. It is possible that codon usageand tRNA acceptor concentrations have coevolved, and that the selectionpressure for this co-evolution is more pronounced for highly expressedgenes than genes expressed at low levels.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,G/T cassette) is used for subjecting each original codon in aparental polynucleotide template to a full range of codon substitutions.In another aspect, at least two degenerate N,N,G/T cassettes areused—either in the same oligo or not, for subjecting at least twooriginal codons in a parental polynucleotide template to a full range ofcodon substitutions. Thus, more than one N,N,G/T sequence can becontained in one oligo to introduce amino acid mutations at more thanone site. This plurality of N,N,G/T sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In another aspect, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,G/T sequence, to introduce any combinationor permutation of amino acid additions, deletions, and/or substitutions.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,G/T sequence.For example, it may be desirable in some instances to use (e.g., in anoligo) a degenerate triplet sequence comprised of only one N, where saidN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence.

It is appreciated, however, that the use of a degenerate N,N,G/T tripletas disclosed herein is advantageous for several reasons. In one aspect,this invention provides a means to systematically and fairly easilygenerate the substitution of the full range of possible amino acids (fora total of 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the instantinvention provides a way to systematically and fairly easily generate2000 distinct species (i.e., 20 possible amino acids per position X 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T triplet, 32individual sequences that code for 20 possible amino acids. Thus, in areaction vessel in which a parental polynucleotide sequence is subjectedto saturation mutagenesis using one such oligo, there are generated 32distinct progeny polynucleotides encoding 20 distinct polypeptides. Incontrast, the use of a non-degenerate oligo in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.

Thus, in a preferred embodiment, each saturation mutagenesis reactionvessel contains polynucleotides encoding at least 20 progeny polypeptidemolecules such that all 20 amino acids are represented at the onespecific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a change in property (when comparedto the template polypeptide), it can be sequenced to identify the aminoacid substitution responsible for such change contained therein.

The template polypeptide may be any protein, however proteins which havea convenient assay for activity such as catalytic activity or ligandbinding are preferred. As used herein, a ligand is any molecule whichbinds specifically to a larger one, such as small molecule binding to aprotein. Representative examples of target interactions includecatalysis, enzyme-substrate interactions, protein-nucleic acidinteractions, receptor-ligand interactions, protein-metal interactionsand antibody-antigen interactions. Representative target proteinsinclude enzymes, antibodies, cytokines, receptors, DNA binding proteins,chelating agents, and hormones.

Any chemical synthetic or recombinant mutagenic method may be used togenerate the population of mutant polypeptides. The practice of thepresent invention may employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal., U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Cabs eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymnology, Vols. 154 and155 (Wu et al. eds), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embiyo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In one embodiment, the template polypeptide is an antibody. The antibodyis subjected to the methods described herein to, for example, map andunderstand which positions within the CDR effect binding affinity. Thetechniques for preparing and using various antibody-based constructs andfragments thereof are well known in the art. An important aspect of thepresent invention is the identification of residues that play, or arelikely to play, a role in the interaction of interest (e.g.,antigen-antibody interaction, metal chelation, receptor binding,substrate binding, etc). Any antibody or antibody fragment may be usedaccording to the present invention.

In one embodiment, any of the evolution platforms CPE, CPI, CPD and CPScan be utilized for generating agonist antibodies, i.e. activatingantibodies. These evolution technologies enable the generation ofagonist antibodies beyond simpler protein crosslinking type activationand in particular allow the activation of receptors such as GPL-1 or 2that are traditionally activated by peptides.

In one aspect, antibodies are selected by FACS or microscopy orequivalent for weakly activating antibodies by using cells withfluorescent signals that fluoresce when the cell surface receptor isactivated. Subsequently, the evolution tools are used to enhance thisactivation. The CPS technology is then utilized to combine up-mutants.

In another aspect, an antibody is selected that binds the receptoractivation site as determined by epitope mapping. CPE, CPI and/or CPDtechniques are used to select for mutants that cause stimulation of thereceptor as determined by an intracellular read-out such as fluorescencein response to calcium ion release or other assays that are well knownin the art. The CPS technology is then utilized to combine up-mutants.

In a particular aspect, some of the key advantages of CPI with single,double or triple amino acid insertions are that these inserted aminoacids can extend into the binding pocket of the receptor to activate thereceptor. In another particular aspect, CPD can remodel and/orreposition amino acids interacting with the receptor to improve oreffect activation and finally CPE can perform relatively smaller changesto effect receptor activation.

The specificity of an antibody is determined by the complementaritydetermining regions (CDRs) within the light chain variable regions (VL)and heavy chain variable regions (VH). The Fab fragment of an antibody,which is about one-third the size of a complete antibody contains theheavy and light chain variable regions, the complete light chainconstant region and a portion of the heavy chain constant region. Fabmolecules are stable and associate well due to the contribution of theconstant region sequences. However, the yield of functional Fabexpressed in bacterial systems is lower than that of the smaller Fvfragment which contains only the variable regions of the heavy and lightchains. The Fv fragment is the smallest portion of an antibody thatstill retains a functional antigen binding site. The Fv fragment has thesame binding properties as the Fab, however without the stabilityconferred by the constant regions, the two chains of the Fv candissociate relatively easily in dilute conditions.

In one aspect, VH and VL regions can be fused via a polypeptide linker(Huston et al., 1991) to stabilize the antigen binding site. This singlepolypeptide Fv fragment is known as a single chain antibody (scFv). TheVH and VL can be arranged with either domain first. The linker joins thecarboxy terminus of the first chain to the amino terminus of the secondchain.

One of skill in the art will recognize that single chain Fv, heavy orlight chain Fv or Fab fragments may be used with this system. A heavy orlight chain can be mutagenized followed by the addition of thecomplementary chain to the solution. The two chains are then allowed tocombine and form a functional antibody fragment. Addition of randomnon-specific light or heavy chain sequences allows for the production ofa combinatorial system to generate a library of diverse members.

Generally, a single-chain expression polynucleotide is generated. Thisexpression polynucleotide contains: (1) a single-chain antibody cassetteconsisting of a V_(H) domain, spacer peptide, and V_(L) domain operablylinked to encode a single-chain antibody, (2) a promoter suitable for invitro transcription (e.g., T7 promoter, SP6 promoter, and the like)operably linked to ensure in vitro transcription of the single-chainantibody cassette forming a mRNA encoding a single-chain antibody, and(3) a transcription termination sequence suitable for functioning in anin vitro transcription reaction. Optionally, the expressionpolynucleotide may also comprise an origin of replication and/or aselectable marker. An example of a suitable expression polynucleotide ispLM166.

The V_(H) and V_(L) sequences can be conveniently obtained from alibrary of V_(H) and V_(L) sequences produced by PCR amplification usingV gene family-specific primers or V gene-specific primers (Nicholls etal., J. Immunol. Meth., 1993, 165: 81; W093/12227) or are designedaccording to standard art-known methods based on available sequenceinformation. Typically, mouse or human V_(H) and V_(L) sequences areisolated. The V_(H) and V_(L) sequences are then ligated, usually withan intervening spacer sequence (e.g., encoding an in-frame flexiblepeptide spacer), forming a cassette encoding a single-chain antibody.Typically, a library comprising a plurality of V_(H) and V_(L) sequencesis used (sometimes also with a plurality of spacer peptide speciesrepresented), wherein the library is constructed with one or more of theV_(H) and V_(L) sequences mutated to increase sequence diversityparticularly at CDR residues, sometimes at framework residues. V regionsequences can be conveniently cloned as cDNAs or PCR amplificationproducts for immunoglobulin-expressing cells. For example, cells fromhuman hybridoma, or lymphoma, or other cell line that synthesizes eithercell surface or secreted immunoglobulin may be used for the isolation ofpolyA+ RNA. The RNA is then used for the synthesis of oligo dT primedcDNA using the enzyme reverse transcriptase (for general methods see,Goodspeed et al., Gene 1989, 76: 1; Dunn et al., J. Biol. Chem., 1989,264: 13057). Once the V-region CDNA or PCR product is isolated, it iscloned into a vector to form a single-chain antibody cassette.

To accomplish construction of antibodies and antibody fragments, theencoding genes are isolated and identified. The genes can be modified topermit cloning into an expression vector or an in vitrotranscription/translation. Although methods can be used such as probingthe DNA for VH and VL from hybridoma cDNA (Maniatis et al., 1982) orconstructing a synthetic gene for VH and VL (Barbas et al., 1992), aconvenient mode is to use template directed methods to amplify theantibody sequences. A diverse population of antibody genes can beamplified from a template sample by designing primers to the conservedsequences at the 3′ and 5′ ends of the variable region known as theframework or to the constant regions of the antibody (Iverson et al.,1989). Within the primers, restriction sites can be placed to facilitatecloning into an expression vector. By directing the primers to theseconserved regions, the diversity of the antibody population ismaintained to allow for the construction of diverse libraries. Thespecific species and class of antibody can be defined by the selectionof the primer sequences as illustrated by the large number of sequencesfor all types of antibodies given in Kabat et al., 1987, herebyincorporated by reference.

Messenger RNA isolated from the spleen or peripheral blood of an animalcan be used as the template for the amplification of an antibodylibrary. In certain circumstances, where it is desirable to display ahomogeneous population of antibody fragments on the cell surface, mRNAmay be isolated from a population of monoclonal antibodies. MessengerRNA from either source can be prepared by standard methods and useddirectly or for the preparation of a CDNA template. Generation of mRNAfor cloning antibody purposes is readily accomplished by following thewell-known procedures for preparation and characterization of antibodies(see, e.g., Antibodies: A Laboratory Manual, 1988; incorporated hereinby reference).

Generation of monoclonal antibodies (MAbs) follows generally the sameprocedures as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance and collecting antisera from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or agoat. Because of the relatively large blood volume of rabbits, rabbitsare usually preferred for production of polyclonal antibodies.

Immunogenic compositions often vary in immunogenicity. It is oftennecessary therefore to boost the host immune system, as may be achievedby coupling a peptide or polypeptide immunogen to a carrier. Exemplaryand preferred carriers are keyhole limpet hemocyanin (KLH) and bovineserum albumin (BSA). Other albumins such as ovalbumin, mouse serumalbumin or rabbit serum albumin can also be used as carriers. Recognizedmeans for conjugating a polypeptide to a carrier protein are well knownand include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimideester, carbodiimides and bis-diazotized benzidine.

The immunogenicity of a particular immunogen composition may be enhancedby the use of non-specific stimulators of the immune response, known asadjuvants. Exemplary and preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated, stored and thespleen harvested for the isolation of mRNA from the polyclonal responseor the animal can be used to generate MAbs for the isolation of mRNAfrom a homogeneous antibody population.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g. a small moleculehapten conjugated to a carrier, a purified or partially purifiedprotein, polypeptide or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are frequently used animals;however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, pp. 60-61, 1986), butmice are preferred, particularly the BALB/c mouse as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from blood samples. Spleencells and blood cells are preferable, the former because they are a richsource of antibody-producing cells that are in the dividing plasmablaststage, and the latter because blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×107 to 2×108lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, 1984). Forexample, where the immunized animal is a mouse, one may use P3-X63/Ag8,X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3,IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 areall useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., 1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. Simple and rapid assaysinclude radioimmunoassays, enzyme immunoassays, cytotoxicity assays,plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are serially diluted and cloned into individualantibody-producing cell lines from which clones can then be propagatedindefinitely to provide MAbs. The cell lines may be exploited for MAbproduction in two basic ways. A sample of the hybridoma can be injected(often into the peritoneal cavity) into a histocompatible animal of thetype that was used to provide the somatic and myeloma cells for theoriginal fusion. The injected animal develops tumors secreting thespecific monoclonal antibody produced by the fused cell hybrid. The bodyfluids of the animal, such as serum or ascites fluid, can then be tappedto provide MAbs in high concentration. The individual cell lines couldalso be cultured in vitro, where the MAbs are naturally secreted intothe culture medium from which they can be readily obtained in highconcentrations. MAbs produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography.

Following the isolation and characterization of the desired monoclonalantibody, the mRNA can be isolated using techniques well known in theart and used as a template for amplification of the target sequence.

A number of template dependent processes are available to amplify thetarget sequences before and after mutagenesis. One of the best knownamplification methods is the polymerase chain reaction (referred to asPCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202and 4,800,159, and in Innis et al., 1990, each of which is incorporatedherein by reference in its entirety. Briefly, in PCR, two primersequences are prepared which are complementary to regions on oppositecomplementary strands of the target sequence. An excess ofdeoxynucleoside triphosphates are added to a reaction mixture along witha DNA polymerase, e.g., Taq polymerase. If the target sequence ispresent in a sample, the primers will bind to the target and thepolymerase will cause the primers to be extended along the targetsequence by adding on nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction products and the process isrepeated. Preferably a reverse transcriptase PCR amplification proceduremay be performed in order to quantify the amount of target amplified.Polymerase chain reaction methodologies are well known in the art. Usingenzymatic amplification techniques such as PCR, desired control elementsmay be designed into the primer and thus, will be incorporated into theDNA product.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method. In this method, a replicativesequence of RNA which has a region complementary to that of a target isadded to a sample in the presence of an RNA polymerase. The polymerasewill copy the replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids (Walker et al., 1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR) involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having a 3′ and 5′ sequences of non-specific DNA and middlesequence of specific RNA is hybridized to DNA which is present in asample. Upon hybridization, the reaction is treated with RNaseH, and theproducts of the probe identified as distinctive products which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Other amplification methods are described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR like, template and enzyme dependentsynthesis. The primers may be modified by labeling with a capture moiety(e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latterapplication, an excess of labeled probes is added to a sample. In thepresence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labeled probe signals thepresence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989). In NASBA, the nucleicacids can be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double-stranded by addition of secondtarget specific primer, followed by polymerization. The double strandedDNA molecules are then multiply transcribed by a polymerase such as T7or SP6. In an isothermal cyclic reaction, the RNAs are reversetranscribed into double stranded DNA, and transcribed once against witha polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Kienow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “race” and “one-sidedPCR” (Frohman, 1990; O'Hara et al., 1989).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, alsomay be used in the amplification step (Wu et al., 1989).

Amplification products may be analyzed by agarose, agarose-acrylamide orpolyacrylamide gel electrophoresis using standard methods (see, e.g.,Maniatis et al., 1982). For example, one may use a 1% agarose gelstained with ethidium bromide and visualized under UV light.Alternatively, the amplification products may be integrally labeled withradio- or fluorometrically-labeled nucleotides. Gels can then be exposedto x-ray film or visualized under the appropriate stimulating spectra,respectively.

Mutagenic procedures of the present invention may comprise any mutagenicapproach that may be tailored to a particular site in a gene, i.e.,site-directed or site-specific mutagenesis. Because the presentinvention relies on comprehensive mutagenesis, the present inventioncontemplates as preferred embodiments those mutagenic procedures thatare rapid, efficient and cost effective.

In one embodiment, the mutagenic procedure utilizes chemical synthesistechniques. In so doing, it is possible to exactly place thesubstitution at one or more particular locations within the gene, andalso to specifically define the nature of the alterations. Chemicalsynthesis methods for DNA are well known within the art. Solid phasetechniques are preferred in this regard.

One advantage to the solid phase method of gene synthesis is theopportunity for mutagenesis using combinatorial synthesis techniques.Combinatorial synthesis techniques are defined as those techniquesproducing large collections or libraries of compounds simultaneously, bysequentially linking different building blocks. Libraries can beconstructed using compounds free in solution, but preferably thecompound is linked to a solid support such as a bead, solid particle oreven displayed on the surface of a microorganism.

Several methods exist for combinatorial synthesis (Holmes et al., 1995;Burbaum et al., 1995; Martin et al., 1995; Freier et al., 1995; Pei etal., 1991; Bruce et al., 1995; Ohlmeyer et al., 1993), including splitsynthesis or parallel synthesis. Split synthesis may be used to producesmall amounts of a relatively large number of compounds, while parallelsynthesis will produce larger amounts of a relatively small number ofcompounds. In general terms, using split synthesis, compounds aresynthesized on the surface of a microparticle. At each step, theparticles are partitioned into several groups for the addition of thenext component. The different groups are then recombined and partitionedto form new groups. The process is repeated until the compound iscompleted. Each particle holds several copies of the same compoundallowing for facile separation and purification. Split synthesis canonly be conducted using a solid support.

An alternative technique known as parallel synthesis may be conductedeither in solid phase or solution. Using parallel synthesis, differentcompounds are synthesized in separate receptacles, often usingautomation. Parallel synthesis may be conducted in microtiter platewhere different reagents can be added to each well in a predefinedmanner to produce a combinatorial library. Parallel synthesis is thepreferred approach for use with enzymatic techniques. It is wellunderstood that many modifications of this technique exist and can beadapted for use with the present invention. Using combinatorial methods,a large number of mutant gene templates may be synthesized.

Mutants genes also may be generated by semisynthetic methods known inthe art (Barbas et al., 1992). Using the conserved regions of anantibody fragment as a framework, variable regions can be inserted inrandom combinations one or more at a time to alter the specificity ofthe antibody fragment and generate novel binding sites, especially inthe generation of antibodies to antigens not conducive to immunizationsuch as toxic or labile compounds. Along the same lines, a knownantibody sequence may be varied by introducing mutations randomly. Thismay be accomplished by methods well known in the art such as the use oferror-prone PCR.

Using the appropriate oligonucleotide primers, PCR is used for the rapidsynthesis of the DNA template containing one or more mutations in thebinding protein gene. Site-specific mutagenesis is a technique useful inthe preparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site-specific mutagenesis allows the production ofmutants through the use of specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Typically, a primer ofabout 17 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

In certain applications, substitution of amino acids by site-directedmutagenesis, it is appreciated that lower stringency conditions arerequired. Under these conditions, hybridization may occur even thoughthe sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated, and thus will generally be a method of choice depending onthe desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl2, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

In a particular embodiment, overlap PCR may be employed. Briefly, aplasmid is used as a template for the first round of PCR. The PCRproducts from the first round are purified and used, together withoutside primers, in the overlap extension PCR reaction. The end productscontained the site directed replacement of a given amino acid with allother possible amino acid residues.

The mutagenized DNA template for the polypeptide of interest can becloned into a plasmid for in vitro transcription/translation or in thepreferred embodiment, the appropriate control elements are includedwithin the PCR product for direct in vitro transcription/translation. Invitro transcription/translation of genes uses cell free extracts toprovide the required enzymes, ribosomes and protein factors. T hesynthesis of proteins is directed by mRNA synthesized from the desiredDNA templates. The DNA template must contain the appropriate controlelements for the system used including a ribosome binding site andpromoter sequence. One of skill in the art would clearly recognize theappropriate required elements for each system.

Prokaryotic in vitro techniques for protein production were the first tobe used (Zubay et al., 1970). Subsequently eukaryotic systems weredeveloped using wheat germ (Roberts, 1973) and rabbit reticulocytes(Pelham, 1976). Several new developments have increased the efficiencyof these techniques. Examples include, the development of nucleasedeficient strains of E. coli to improve the results using linear DNAtemplates (Yang, 1980) and treatment of reticulocyte lysates withmicrococcal nuclease to lower any background expression from the system.

The most recent systems developed for in vitro transcription/translationare based on transcription by phage RNA polymerases including SP6 andSP7 (Krieg, 1987, Studier, 1990). DNA placed under the control of T7promoter elements can be used as a template for in vitro transcriptionby T7 RNA polymerase or for complete in vitro transcription/translationwith the polymerase added to either a prokaryotic or eukaryotic proteinsynthesis system. While the methods of the present invention can be usedwith any in vitro transcription/translation system, the T7 system ispreferred for transcription and the use of a prokaryotic translationsystem is preferred as no capping of the RNA is required.

Using in vitro methods for translation, amino acid derivatives may beincorporated into the protein by addition of the derivatized amino acidto the protein synthesis system mixture. Varying the concentration ofthe derivatives, with respect to the normal amino acid, permits one tocreate a mixed population and measure relative effects.

Mutant polypeptides generated by the present invention may becharacterized using a variety of techniques. In general, proteinproducts may be analyzed for the correct apparent molecular weight usingSDS-PAGE. This provides an initial indication that the polypeptide was,in fact, synthesized. When compared to the natural molecule, it alsoindicates whether normal folding or processing is taking place with themutant. In this regard, it may prove useful to label the polypeptide.Alternatively, the polypeptide may be identified by staining of the gel.

Beyond mere synthesis, proteins may be characterized according tovarious properties and an extensive range of functions. Propertiesinclude isoelectric point, thermal stability, sedimentation rate andfolding. One manner of examining folding is the ability to be recognizedby a cognate binding partner. The prime example of this function is theantibody-antigen interaction. A wide variety of different immunoassayformats are available for this purpose and are well known in the art.Principally, changes in either affinity or specificity can be determinedwhen the protein is contacted with a specific ligand or panels ofrelated ligands.

Immunoassays can be generally divided into two types: heterogeneousassays requiring multiple separation steps, and homogeneous assays whichare performed directly. Heterogeneous immunoassays in general involve aligand or antibody immobilized on a solid matrix. A sample containing aligand is contacted with the immobilized antibody and the amount ofcomplex formed on the matrix support is determined from a label attacheddirectly or indirectly to the immobilized complex. As used in thecontext of the present invention, ligand is defined as a species thatinteracts with a non-identical molecule to form a tightly bound, stablecomplex. For practical purposes, the binding affinity is usually greaterthan about 106 M⁻¹ and is preferably in the range of 10⁹-10′^(s) M⁻¹.The ligand may be any of several types of organic molecules, includingalicyclic hydrocarbons, polynuclear aromatics, halogenated compounds,benzenoids, polynuclear hydrocarbons, nitrogen heterocyclics, sulfurheterocyclics, oxygen heterocyclics, and alkane, alkene alkynehydrocarbons, etc. Biological molecules are of particular interest,including amino acids, peptides, proteins, lipids, saccharides, nucleicacids and combinations thereof. Of course it will be understood thatthese are by way of example only and that contemplated immunoassaymethods are applicable to detecting an extraordinarily wide range ofcompounds, so long as one can obtain an antibody that binds with theligand of interest.

Heterogeneous immunoassays may be performed as sandwich assays in whicha molecule of interest is reacted with an immobilized antibody thatspecifically binds that molecule with high affinity. In a second step, aconjugate formed from the same or different antibody to the antigen anda marker molecule is reacted with the antigen-antibody complex on theimmobilization matrix. After removal of excess free marker conjugate,the bound marker conjugate, which is proportional to the amount ofligand in the sample, is measured.

Detection of immunocomplex formation is well known in the art and may beachieved through the application of numerous approaches. Theseapproaches are typically based upon the detection of a label or marker,such as any of the radioactive, fluorescent, chemiluminescent,electrochemiluminescent, biological or enzymatic tags or labels known inthe art. U.S. Patents concerning the use of such labels include U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241, each incorporated herein by reference. Ofcourse, one may find additional advantages through the use of asecondary binding ligand such as a second antibody or a biotin/avidinligand binding arrangement, as is known in the art.

Preferred methods for detection includes radioimmunoassay (RIA) orenzyme-linked immunosorbent assay (ELISA) with ELISA being mostpreferred due to generally increased sensitivity. ELISAs are extensivelyused in biotechnology applications, particularly as immunoassays for awide range of antigenic substances. The sensitivity of ELISA is based onthe enzymatic amplification of the signal

Other preferred proteins contemplated for use in accordance with thepresent invention are those which have a convenient assay for activity.Representative examples of target interactions include catalysis,enzyme-substrate interactions, protein-nucleic acid interactions,receptor-ligand interactions and protein-metal interactions. In theseassays the mutant proteins can be compared with the wild-type proteinfor changes in the ability to perform any of the foregoing functions.

As used herein, the term “contacting” is defined as bringing thereaction components into close enough proximity to each other to allowthe desired interaction to occur. Contacting may be accomplished bymixing the components in solution, for example, or by heterogeneousinteraction such as by flow contact through a column or immobilizingmatrix that binds to one of the components.

For mutant proteins having a catalytic activity, the appropriatereaction may be monitored for a change in catalytic rate or analteration in specificity.

The antibodies produced and isolated by the method of the invention areselected to bind a predetermined target. Typically, the predeterminedtarget will be selected in view of its applicability as a diagnosticand/or therapeutic target. The predetermined target may be a known orunknown epitope Antibodies generally bind to a predetermined antigen(e.g., the immunogen) with an affinity of about at least 1×10⁷ M^(−1,)preferably with an affinity of about at least 5×10⁷ M⁻¹ more preferablywith an affinity of at least 1×10⁸ M-1 to 1×10⁹ M⁻¹ or more, sometimesup to 1×10¹⁰ M⁻¹ or more. Frequently, the predetermined antigen is ahuman protein, such as for example a human cell surface antigen (e.g.,CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other humanbiological macromolecule (e.g., thrombomodulin, protein C, carbohydrateantigen, sialyl Lewis antigen, L-selectin), or nonhuman diseaseassociated macromolecule (e.g., bacterial LPS, virion capsid protein orenvelope glycoprotein) and the like.

In another example, several reports of the diagnostic and therapeuticutility of scFv have been published (Gruber et al., 1994 op.cit.; Lilleyet al., 1994 op.cit.; Huston et al., Int. Rev. Immunol 1993, 10:a 195,Sandhu JS, Crit. Rev. Biotechnol., 1992,12: 437).

High affinity antibodies of the desired specificity can be engineeredand expressed in a variety of systems. For example, scFv have beenproduced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861) andcan be readily made in prokaryotic systems (Owens R J and Young R J, J.Immunol. Meth., 1994,168: 149; Johnson S and Bird R E, Methods Enzymol.,1991, 203: 88). Furthermore, the single-chain antibodies can be used asa basis for constructing whole antibodies or various fragments thereof(Kettleborough et al., Euro J. Immunol., 1994, 24: 952). The variableregion encoding sequence may be isolated (e.g., by PCR amplification orsubcloning) and spliced to a sequence encoding a desired human constantregion to encode a human sequence antibody more suitable for humantherapeutic uses where immunogenicity is preferably minimized. Thepolynucleotide(s) having the resultant fully human encoding sequence(s)can be expressed in a host cell (e.g., from an expression vector in aeukaryotic cell) and purified for pharmaceutical formulation.

The DNA expression constructs will typically include an expressioncontrol DNA sequence operably linked to the coding sequences, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences will be eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells.Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences, and the collection and purification of themutant “engineered” antibodies.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to an expression controlsequence (i.e., positioned to ensure the transcription and translationof the structural gene). These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors willcontain selection markers, e.g., tetracycline or neomycin, to permitdetection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

In addition to eukaryotic microorganisms such as yeast, mammalian tissuecell culture may also be used to produce the polypeptides of the presentinvention (see, Winnacker, “From Genes to Clones,” VCH Publishers, N.Y.,N.Y. (1987), which is incorporated herein by reference). Eukaryoticcells are preferred, because a number of suitable host cell linescapable of secreting intact immunoglobulins have been developed in theart, and include the CHO cell lines, various COS cell lines, HeLa cells,myeloma cell lines, B-cells or hybridomas. Expression vectors for thesecells can include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al., Immunol. Rev. 1986,89: 49), and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes,cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.

Eukaryotic DNA transcription can be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting sequences of between10 to 30 obp that increase transcription by a promoter. Enhancers caneffectively increase transcription when either 5′ or 3′ to thetranscription unit. They are also effective if located within an intronor within the coding sequence itself. Typically, viral enhancers areused, including SV40 enhancers, cytomegalovirus enhancers, polyomaenhancers, and adenovirus enhancers Enhancer sequences from mammaliansystems are also commonly used, such as the mouse immunoglobulin heavychain enhancer.

Mammalian expression vector systems will also typically include aselectable marker gene. Examples of suitable markers include, thedihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), orprokaryotic genes conferring drug resistance. The first two marker genesprefer the use of mutant cell lines that lack the ability to growwithout the addition of thymidine to the growth medium. Transformedcells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment.lipofection, or electroporation may be used for other cellular hosts.Other methods used to transform mammalian cells include the use ofPolybrene, protoplast fusion, liposomes, electroporation, andmicroinjection (see, generally, Sambrook et al., supra).

Once expressed, the antibodies, individual mutated immunoglobulinchains, mutated antibody fragments, and other immunoglobulinpolypeptides of the invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,fraction column chromatography, gel electrophoresis and the like (see,generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y.(1982)). Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically or in developing andperforming assay procedures, immunofluorescent stainings, and the like(see, generally, Immunological Methods, Vols. I and II, Eds. Lefkovitsand Pernis, Academic Press, N.Y. N.Y. (1979 and 1981)).

The oligopeptides of the present invention can be used for diagnosis andtherapy. By way of illustration and not limitation, antibodies can beused to treat cancer, autoimmune diseases, or viral infections. Fortreatment of cancer, the antibodies will typically bind to an antigenexpressed preferentially on cancer cells, such as erbB-2, CEA, CD33, andmany other antigens well known to those skilled in the art. Fortreatment of autoimmune disease, the antibodies will typically bind toan antigen expressed on T-cells, such as CD4, the IL-2 receptor, thevarious T-cell antigen receptors and many other antigens well known tothose skilled in the art (e.g., see Fundamental Immunology, 2nd ed., W.E. Paul, ed., Raven Press: New York, N.Y., which is incorporated hereinby reference). For treatment of viral infections, the antibodies willtypically bind to an antigen expressed on cells infected by a particularvirus such as the various glycoproteins (e.g., gB, gD, gE) of herpessimplex virus and cytomegalovirus, and many other antigens well known tothose skilled in the art (e.g., see Virology, 2nd ed., B. N. Fields etal., eds., (1990), Raven Press: New York, N.Y.).

Pharmaceutical compositions comprising antibodies of the presentinvention are useful for parenteral administration, i.e.,subcutaneously, intramuscularly or intravenously. The compositions forparenteral administration will commonly comprise a solution of theantibody or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.4% saline, 0.3% glycine and thelike. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of the mutant antibodies in these formulations can varywidely, i.e., from less than about 0.01%, usually at least about 0.1% toas much as 5% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intramuscular injectioncould be made up to contain 1 ml sterile buffered water, and about 1 mgof mutant antibody. A typical composition for intravenous infusion canbe made up to contain 250 ml of sterile Ringer's solution, and 10 mg ofmutant antibody. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in, for example, Remington's PharmaceuticalScience, 20th Ed., Mack Publishing Company, Easton, Pa. (2000), which isincorporated herein by reference.

Manufacturing of Candidates and/or Evolved Candidates

In one embodiment, the eukaryotic system is a mammalian system selectedfrom one of the group consisting of CHO, HEK293, IM9, DS-1, THP-1, HepG2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116,Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P,BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F, and SP2/0 celllines; and mouse splenocytes and rabbit PBMC.

In one embodiment, a variety of mammalian host cells can be used in themanufacturing of candidate, including Fibroblast cells (3T3, mouse;BHK21, Syrian hamster) Epithelial cells (MDCK, dog; Hela, human; PtK1,rat kangaroo) Plasma cells ((SP2/0 and NS0, mouse) Kidney cells (293,human; COS, monkey) Ovary cells (CHO, Chinese hamster) Embryonic cells(R1 and E14.1, mouse; H1 and H9, human; PER C.6, human).

In certain aspects, the recombinant antibodies are produced in CHO andNS0 and SP2/0 cell lines. In a specific aspect, the mammalian system isa CHO-S cell line. Expression vector systems most frequently used areglutamine synthetase expression systems and others based onDihydrofolate reductase genes.

In another embodiment, the eukaryotic system is a yeast cell system. Inone aspect, the yeast cell system is selected from S. cerevisiae orpicchia cells.

In the method of the present invention, hosts used for screening evolvedmolecules are the same as hosts used for downstream manufacturing ofhits. In another aspect of the present invention, the genetic systemused for discovery and evolution of proteins is exactly the same as thegenetic system used for manufacturing the protein for commercialapplications.

Biosimilars

Biosimilars are protein based therapeutics that have an identical aminoacid sequence (i.e. chemical composition) as an approved ethical drugwhich is no longer patent protected. In one aspect, the CIAO method isparticularly relevant for biosimilars. While it is essential to producethe protein therapeutic in an equivalent formulation and composition, tobe competitive in the marketplace the biosimilar should be made quicklyand as cheaply as possible. Cell culture media and process developmentare some of the most costly and time consuming parts of preparing andproducing a biosimlar.

Changing the silent mutation codons within a protein therapeutic changesthe codon used for protein translation but preserve the amino acidsequence within the protein. These codon changes at a variety ofpositions within a molecule, particularly in the amino terminus can havesignificant impact on expression and in some cases even glycosylation.In one aspect, the CIAO method is used to select and evolve the silentmutation codons in a protein within a host cell similar to the oneultimately used for manufacturing. Therefore processing time can bereduced due to the higher protein yields and the fact that the proteinwas selected to express in the host cell line, so most traditionalmanufacturing issues have been selected out of the molecule. Further, byselecting the molecules in inexpensive, serum free culture media,molecules can be selected with codons that permit inexpensivemanufacturing and purification.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are to be consideredillustrative and thus are not limiting of the remainder of thedisclosure in any way whatsoever.

Antibodies produced as described above may be subjected to one or more“biological activity” assays to select an antibody with beneficialproperties from a therapeutic perspective.

Preparation of the Formulation

After preparation of the antibody of interest, the pharmaceuticalformulation comprising it can be prepared. The antibody to be formulatedcan optionally be subjected to prior lyophilization, or the formulationof interest can be an aqueous formulation. A therapeutically effectiveamount of antibody present in the formulation can be determined bytaking into account the desired dose volumes and mode(s) ofadministration, for example. From about 0.1 mg/mL to about 50 mg/mL,preferably from about 0.5 mg/mL to about 25 mg/mL and most preferablyfrom about 2 mg/mL to about 10 mg/mL is an exemplary antibodyconcentration in some formulations.

An aqueous formulation can be prepared comprising the antibody in apH-buffered solution The buffer can have a pH in the range, for example,from about 4.5 to about 6.0. Examples of buffers that can control the pHwithin this range include acetate (e.g. sodium acetate), succinate (suchas sodium succinate), gluconate, histidine, citrate and other organicacid buffers. The buffer concentration can be from about 1 mM to about50 mM, preferably from about 5 mM to about 30 mM, depending, forexample, on the buffer and the desired isotonicity of the formulation.

A polyol, which may stabilize the antibody, can be included in theformulation. In some cases, the formulation does not contain an amountof a salt, such as sodium chloride, that may cause the antibody toprecipitate and/or may result in oxidation at low pH. In some cases, thepolyol is a nonreducing sugar, such as sucrose or trehalose. The polyolis added to the formulation in an amount which may vary with respect tothe desired isotonicity of the formulation. In some cases, the aqueousformulation is isotonic, in which case suitable concentrations of thepolyol in the formulation are in the range from about 1% to about 15%w/v, for example. However, hypertonic or hypotonic formulations may alsobe suitable. The amount of polyol added may also alter with respect tothe molecular weight of the polyol. For example, a lower amount of amonosaccharide (e g mannitol) may be added, compared to a disaccharide(such as trehalose).

A surfactant can also be added to the antibody formulation. Exemplarysurfactants include nonionic surfactants such as polysorbates (e.g.polysorbates 20, 80 etc) or poloxamers (e.g. poioxamer 188). The amountof surfactant added is such that it reduces aggregation of theformulated antibody and/or minimizes the formation of particulates inthe formulation and/or reduces adsorption. For example, the surfactantmay be present in the formulation in an amount from about 0 001% toabout 0 5%, or from about 0 005% to about 02% or from about 0 01% toabout 0 1%.

Formulations can contain the above-identified agents (/ e antibody,buffer, polyol and surfactant) and may be essentially free of one ormore preservatives, such as benzyl alcohol, phenol, m-cresol,chlorobutanol and benzethonium Cl. Althernatively, a preservative may beincluded in the formulation, particularly where the formulation is amultidose formulation. The concentration of preservative may be in therange from about 0 1% to about .2%, or from about 0 5% to about 1%. Oneor more other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington's PharmaceuticalSciences 16th edition, Osol, A Ed (1980) may be included in theformulation provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include additional buffering agents, co-solvents,antioxidants including ascorbic acid and methionine, chelating agentssuch as EDTA, metal complexes (e g Zn-protem complexes), biodegradablepolymers such as polyesters, and/or salt-forming countenons such assodium.

The formulation may also contain more than one protein as necessary forthe particular indication being treated, preferably those withcomplementary activities that do not adversely affect the other protein.Such proteins are suitably present in combination in amounts that areeffective for the purpose intended.

The formulations to be used for in vivo administration must be sterileThis is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation.

Embodiments indicated herein as exemplary or preferred are intended tobe illustrative and not limiting.

Other embodiments of the invention will be apparent to one skilled inthe field such as, for example, considering combinations of theembodiments referenced above, and are contemplated as covered within thescope of the invention herein.

EXAMPLES Example 1 Generation and Screening of an Antibody Library

This example describes the method of generating and screening amammalian cell surface display human antibody library to isolate humanantibodies with binding activity to a target antigen using thecombination of flow cytometric sorting and ELISA.

Library Screening by Flow Cytometric Analysis

-   -   1. Generate human antibody libraries stably integrated in        mammalian cells such as described in Appendix 1.2, 1.3 and 1.4        below.    -   2. Expand stable fully human antibody library clones prior to        flow cytometric analysis.    -   3. On the day of flow cytometric analysis, wash 1×10⁷ cells with        1× PBS    -   4. Detach cell with Detachin cell detachment medium and collect        cells in 1× PBS    -   5. Spin down cells at 3000 rpm for 5 minutes. Remove        supernatant.    -   6. Re-suspend cell pellet in 1 ml of cold 1× PBS and spin at        3000 rpm for 5 minutes.    -   7. Remove supernatant and re-suspend the cell pellet in 500 μl        of 2 μg/ml of purified human 001 protein in cold 1× PBS. In the        event the desire is to screen to mimic downstream formulation        conditions, any or all of the following additional components        may be added:        -   Sodium acetate, sodium succinate, gluconate, hisitidine,            citrate or other organic acid to adjust to the pH range used            in the formulation        -   Polyol such as sucrose or trehalose in the range of 1 to 15%        -   Surfactant such as polysorbates or poloxamers in the range            of concentration such 0.001%-0.5% or 0.005% to 0.2% or 0.01%            to 0.1%        -   Carriers or stabilizers such as ascorbic acid and methionine            (anti-oxidants), EDTA (chelating agents), Zn-protein            complexes (metal complexes), polymers (polyesters)        -   Additional salts such as sodium, calcium or Zinc    -   8. Incubate on ice for 1 hour with occasionally mixing by hand.    -   9. Spin down cells at 3000 rpm for 5 minutes. Remove        supernatant.    -   10. Re-suspend cell pellet in 1 ml of cold 1× PBS and spin at        3000 rpm for 5 minutes.    -   11. Repeat steps 7 and 8.    -   12. Remove supernatant and re-suspend the cell pellet in 500 μl        of 1 μg/ml of rabbit anti-human 001 polyclonal antibody in cold        1× PBS with 10% goat serum.    -   13. Incubate on ice for 30 minute with occasionally mixing by        hand.    -   14. Spin down cells at 3000 rpm for 5 minutes. Remove        supernatant.    -   15. Re-suspend cell pellet in 1 ml of cold 1× PBS and spin at        3000 rpm for 5 minutes.    -   16. Repeat steps 7 and 8.    -   17. Remove supernatant and re-suspend the cell pellet in 500 μl        of goat anti-rabbit antibody conjugate with FITC and goat        anti-human Fc antibody conjugate with pyroerthrin in cold 1× PBS        with 10% goat serum.    -   18. Incubate on ice for 30 minute with occasionally mixing by        hand.    -   19. Spin down cells at 3000 rpm for 5 minutes. Remove        supernatant.    -   20. Re-suspend cell pellet in 1 ml of cold 1× PBS and spin at        3000 rpm for 5 minutes.    -   21. Repeat steps 7 and 8.    -   22. Remove supernatant and re-suspend the cell pellet in 1 ml of        cold 1× PBS with 2% goat serum.    -   23. Proceed with flow cytometric analysis using Dako MoFlo.    -   24. Draw a sort window to include the top 0.1% of total cells in        terms of ratio of PE/FITC fluorescence. Collect cells that fall        within the sort window in 96 well plates with 100 μl of growth        media.

Recovery of Heavy Chain and Light Chain Variable Region Sequences

-   -   1. Expand the clones from 96 well plates to 6 well plates. When        the cells reach 80% confluence in the 6 well plates, proceed to        genomic DNA isolation using Qiagen DNeasy Tissue kit.    -   2. Aspirate off the media from the cells. Add 500 ml of 1× PBS        to each 6 well. Scrap the cells off the plate with sterile pipet        tips. Transfer scrapped cells in PBS to a sterile        micro-centrifuge tube.    -   3. Centrifuge the cells for 5 minutes at 3000 rpm.    -   4. Remove supernatant and re-suspend cell pellet in 200 μl 1×        PBS.    -   5. Add 20 μl proteinase K and 200 μl Buffer AL to the sample,        mix thoroughly by vortexing, and incubate at 56° C. for 10        minutes.    -   6. Add 200 μl ethanol to the sample and mix thoroughly by        vortexing.    -   7. Pipet the mixture from step 6 into a spin column. Centrifuge        at 8000 rpm for one minute. Discard the flow-through.    -   8. Add 500 μl Buffer AW1 and centrifuge for one minute at 8000        rpm. Discard the flow-through.    -   9. Add 500 μl Buffer AW2 and centrifuge for 2 minutes at 14,000        rpm. Discard the flow-through. Centrifuge again for one minute        at 14,000 rpm. Make sure the membrane is completely dry.    -   10. Place the spin column in a sterile micro-centrifuge tube and        pipet 200 μl Buffer AE directly onto the membrane.    -   11. Incubate at room temperature for one minute and centrifuge        for one minute at 8000 rpm to elute the genomic DNA.    -   12. QC the genomic DNA by setting up the following reactions in        1.5 ml micro-centrifuge tubes:

gDNA 5 μl 10× Sample loading buffer 5 μl Total Volume 10 μl Load onto a 0.8% agarose TAE gel with 0.5 μg/ml Ethidium Bromide. Use1kB DNA ladder as standard. Run the gel at 100V for 20-30 minutes in 1×TAE buffer.

-   -   13. Set up the following PCR reactions in sterile PCR tubes:

gDNA   1 μl 2× HotStar Taq Master Mix 12.5 μl Variable domain forwardprimer*  0.5 μl Variable domain reverse primer*  0.5 μl H2O 10.5 μlTotal Volume   25 μl *see appendix 1.2

-   -   14. Place the PCR tubes in the thermal cycler and start the        cycling program.

Initial activation step: 15 minutes, 95° C.

3-step cycling

Denaturation: 40 seconds, 94° C.

Annealing: 40 seconds, 55° C.

Extension: 2 minutes, 72° C.

Number of cycles: 30

Final extension step: 10 minutes, 72° C.

-   -   15. QC the PCR reactions by setting up the following reactions        in 1.5 ml micro-centrifuge tubes:

PCR reaction 5 μl 10× Sample loading buffer 5 μl Total Volume 10 μl Load onto a 1% agarose TAE gel with 0.5 ug/ml Ethidium Bromide. Use 1kBDNA ladder as standard. Run the gel at 100V for 20-30 minutes in 1× TAEbuffer.

16. Set up the following cloning reactions in 1.5 ml micro-centrifugetubes using Invitrogen TOPO 2.1 kit:

PCR reaction 4 μl Salt Solution 1 μl TOPO vector 1 μl Total Volume 6 μl

-   -   17. Mix reactions gently and incubate for 5 minutes at room        temperature.    -   18. Add 2 μl of the TOPO cloning reaction from step 17 into a        vial of One Shot Chemically competent E. coli and mix gently.    -   19. Incubate on ice for 30 minutes.    -   20. Heat-shock the cells for 30 seconds at 42° C.    -   21. Transfer the tubes to ice and incubate for 2 minutes.    -   22. Add 250 μl of room temperature S.O.C. medium.    -   23. Shake the tubes horizontally at 37° C. for one hour at 200        rpm.    -   24. Spread 10 μl of the transformation on a re-warmed        LB-carbenicillin plate.    -   25. Incubate plate overnight at 37C.    -   26. Pick 6 clones from each transformation for sequencing.    -   27. Analyze the heavy chain and light chain variable region        sequences. Proceed to the second round of screening using the        ELISA method.

Digest vector and Human Antibody Clones with Restriction Enzymes

Reactions will depend on restriction enzyme(s) chosen, and according tomanufacturer's instructions; examples are provided here: Prepare thefollowing digestion reactions in a microcentriftwe tube on ice.

Vector DNA (2 μg) x μl 10× Rest Enz Buffer x 10 μl Nuclease-free waterQS to 97 μl Rest Enz 1(10 U/μl) 3 μl Rest Enz 2 (10 U/μl) 3 μl Totalreaction volume 100 μl

Human antibody clones (5 ug) x μl 10× Rest Enz Buffer x 10 μlNuclease-free water QS to 97 μl Rest Enz 1 (10 U/μl) 3 μl Rest Enz 2 (10U/μl) 3 μl Total reaction volume 100 μl

-   -   1. Mix gently and spin briefly (5 sec.) in microfuge    -   2. Incubate the reaction at 37° C. overnight

CIP digest vector and Purify with QIAquick PCR Purification Kit

-   -   3. Add 2 μl of Apex phosphatase to the microcentrifuge tube        containing the vector digestion reaction.    -   4. Incubate at 37° C. for 10 minutes    -   5. Heat at 70° C. for 5 minutes to inactivate the Apex        phosphatase    -   6. Add 500 μL of Buffer PBI to the microcentrifuge    -   7. Mix by vortexing and quick centrifuge    -   8. Load 750 μL at a time onto a column    -   9. Centrifuge at 12,000×g for 1 minute and decant liquid from        collection tube    -   10. Repeat until all sample has been processed.    -   11. Wash with 750 μL PE Buffer (Ethanol added!)    -   12. Centrifuge at 12, 000×g for 1 minute and decant liquid from        collection tube    -   13. Place column back onto collection tube and centrifuge again    -   14. Put column onto new microcentrifuge tubes and elute with 50        μL EB Buffer.

Gel purify Restriction Enzyme Digested Human Antibody Clones

-   -   1. Set up the following reactions in a 1.5 ml micro-centrifuge        tube:

Rest Enz digested Fully human antibody clones 100 μl 10× Sample loadingbuffer  3 μl Total Volume 103 μl

-   -   2. Load onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium        Bromide. Use 1kB DNA ladder as standard. Run the gel at 100V for        20-30 minutes in 1× TAE buffer.    -   3. Cut out the bands corresponding to the heavy chain (HC) and        light chain (LC) variable regions and purified using QIAquick        Gel Extraction Kit.    -   4. Add 3 volume of buffer QG to 1 volume of gel.    -   5. Incubate at 50° C. for 10 minutes until the gel slice has        completely dissolved. Add 1 gel volume of isopropanol to the        sample and mix.    -   6. Place a QIAquick spin column in a provided 2 ml collection        tube.    -   7. Apply the sample to the QIAquick column, and centrifuge for 1        minute.    -   8. Discard flow-through and place QIAquick column back in the        same collection tube.    -   9. Add 0.75 ml of buffer PE to QIAquick column and centrifuge        for 1 minute.    -   10. Discard the flow-through and centrifuge the QIAquick column        for an additional 1 minute at 17,900×g (13,000 rpm).    -   11. Place QIAquick column into a clean 1.5 ml microcentrifuge        tube.    -   12. Add 52 μl of buffer EB to the center of the QIAquick        membrane and centrifuge the column for 1 minute. Let the column        stand for 1 minute, and then centrifuge for 1 minute.

Ligate Human HC and LC Variable Domain into Digested Vector DNA

Prepare the following ligation reaction in a microcentrifuge tube onice:

Digested vector DNA (100 ng) x μl Human HC and LC variable domain y μl5× T4 ligase Buffer 4 μl Nuclease-free water QS to 19 μl T4 ligase(2,000 U/μl) 1 μl Total reaction volume 20 μl

-   -   1. Mix gently and spin briefly (5 sec.) in microfuge    -   2. Incubate at room temperature for 2 hours or 16° C. overnight    -   3. Transform each of the ligation reaction mixtures into        Supercompetent E. coli cells    -   4. Pre-chill 14 ml BD Falcon polypropylene round-bottom tubes on        ice. Prepare SOC medium to 42° C.    -   5. Thaw the Supercompetent cells on ice. When thawed, gently mix        and aliquot 100u1 of cells into each of the pre-chilled tubes.    -   6. Add 1.7 μl of β-mercaptoethanol to each aliquot of cells.        Incubate the cells on ice for 10 minutes, swirling gently every        2 minutes.    -   7. Add 2 μl of the ligation reaction mixture to one aliquot of        cells. Flick the tubes gently.    -   8. Incubate the tubes on ice for 30 minutes.    -   9. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.    -   10. Incubate the tubes on ice for 2 minutes    -   11. Add 900 μl of preheated SOC medium and incubate the tubes at        37° C. for 1 hour with shaking at 225-250 rpm.    -   12. Plate 20 μl and 200 μl of the transformation mixture on LB        agar plates containing carbenicillin.    -   13. Incubate the plates at 37° C. overnight.    -   14. Count colonies on plates and pick 6 colonies for PCR        screening and sequencing.    -   15. Choose one clone with the correct sequence, prepare plasmid        DNA, and proceed to transfection in 293F cells.

Transfection of 293F Cells

-   -   1. One week before transfection, transfer 293F cells to        monolayer culture in serum supplemented Dulbecco's Modified        Eagle Medium (D-MEM).    -   2. One day before transfection, plate 0.1×10⁵ cells in 100 μl of        serum supplemented D-MEM per transfection sample in 96 well        formats.    -   3. For each transfection sample, prepare DNA-Lipofectamine        complexes.    -   4. Dilute 0.2 μg of DNA in 50 μl Opti-MEM Reduced Serum Medium.        Mix gently.    -   5. Dilute 0.125 μl Lipofecctamine in 50 μl Opti-MEM Reduced        Serum Medium. Mix gently and incubate for 5 mM at room        temperature.    -   6. Combine the diluted DNA with the diluted Lipofectamine Mix        gently and incubate for 20 mM at room temperature.    -   7. Add the 100 μl DNA-Lipofectamine complexes to each well        containing cells and medium. Mix gently by rocking the plate        back and forth.    -   8. Incubate the cells at 37° C. in a 5% CO₂ incubator.    -   9. Add 100 μl of serum supplemented D-MEM to each well after 6        hours. Incubate the cells at 37° C. in a 5% CO₂ incubator        overnight.    -   10. Aspirate off medium in each well. Wash each well with 100 μl        of 293 SFM II with 4 mM L-Glutamine Add 100 μl of 293 SFM II        with 4 mM L-Glutamine to each well.    -   11. Collect supernatant for ELISA at 96 hours after        transfection.

Appendix 1.1: Buffer Recipes

1× PBS with 2% goal serum

-   -   2 ml goat serum    -   98 ml 1× PBS

50× TAE buffer

-   -   242 g Tris base    -   57.1 ml glacial acetic acid    -   37.2 g Na₂EDTA-2H2O    -   Add distilled H₂O to final volume of 1 liter

1× TAE buffer

-   -   20 ml 50× TAE buffer    -   800 ml distilled H₂O

0.8% Agarose Gel with ethidium bromide

-   -   0.8 g LE agarose    -   100 ml 1× TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

1% Agarose Gel with ethidium bromide

-   -   1 g LE agarose    -   100 ml 1× TAE buffer    -   Melt the agarose in a microwave oven and swirl to ensure even        mixing    -   Cool agarose to 55° C.    -   Add 2.5 μl of 20 mg/ml Ethidium Bromide to agarose    -   Pour onto a gel platform

LB

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH    -   Autoclave

LB-carbenicillin agar

-   -   10 g NaCl    -   10 g tryptone    -   5 g yeast extract    -   20 g agar    -   Add distilled H₂O to a final volume of 1 liter    -   Adjust pH to 7.0 with 5 N NaOH

Autoclave

-   -   Cool to 55° C.    -   Add 10 ml of 10 mg/ml of filter-sterilized carbenicillin    -   Pour into petri dishes (25 ml/100-mm plate)

SOC Medium

-   -   0.5 g NaCl    -   20 g tryptone    -   0.5 g yeast extract    -   2 ml of filter-sterilized 20% glucose    -   Add distilled H₂O to a final volume of 1 liter    -   Autoclave    -   Add 10 ml of filter-sterilized 1 M MgCl₂ and 10 ml of        filter-sterilized 1 M MgSO_(s) prior to use

Washing solution

-   -   0.05% Tween-20 in PBS

Blocking solution

-   -   2% Carnation non-fat milk in PBS

Heat inactivated fetal bovine serum

-   -   500 ml heat inactivated fetal bovine serum in the original        vendor bottle    -   Heat for 30 minutes at 56° C. with mixing every 5 minutes    -   Prepare 50 ml aliquots and store at −20° C.

Serum supplemented Dulbecco's Modified Eagle Medium

-   -   500 ml Dulbecco's Modified Eagle Medium    -   50 ml heat inactivated fetal bovine serum    -   5 ml 10 mM MEM Non-Essential Amino Acids

293 SFM II with 4 mM L-Glutamine

-   -   500 ml SFM II    -   10 ml 200 mM L-Glutamine

DEAE-Dextran solution

-   -   1 g DEAE-dextran (diethylaminoethyl-dextran)    -   Dissolve in 100 ml of distilled water    -   Filter sterilize

Appendix 1.2: Construction of Fully Human Antibody Library

All functional human germline heavy chain (V_(H)) and kappa light chain(V_(k)) V regions can be obtained from V base(http://vbase.mrc-cpe.cam.ac.uk/) and aligned. The alignments can thenbe analyzed regarding diversity, especially in the framework threeregions. Desired number of V_(H) and V_(k) genes from the resultingsequence clusters can then be selected for library construction.

V Region Cloning

Heavy and light chain V region genes (including Frameworks 1, 2, and 3and CDR1, CDR2 and CDR3) are amplified from human genomic DNA in twopieces using gene specific primers. Partial V-region genes are thencombined by overlap PCR. A linker is added to full length LC V-regionsby nested PCR before cloning into mammalian expression vector. Cloned LCvariable domains are sequence confirmed (yielding LC V clones). Theheavy chain variable domains are TOPO cloned and sequence confirmed (HCV TOPO clones). Sequence confirmed HC V regions are amplified from thecorresponding plasmids, a linker is added to the 3′ end, and theresulting PCR products are cloned into the LC V variable domain clonesto form Vk/VH combinations and into a mammalian vector.

Expression of Full Length IgGs

Expression of full length kappa light chain and IgG1 heavy chain in thedesired mammalian vector can be driven from a single promoter, forexample, CMV promoter. Each chain is preceded by a secretion signaltargeting the nascent polypeptide chain to the endoplasmatic reticulum(ER). An anchoring signal can be fused to the C-terminus of the heavychain. This signal is cleaved off and replaced with an anchor whichattaches the full length IgG to the outside of the cell membrane aftersecretion.

Appendix 1.3: Generation of Stable Fully Human Antibody Library inMammalian Cells by Transfection

-   -   1. One week before transfection, transfer CHO-S cells to        monolayer culture in serum supplemented Dulbecco's Modified        Eagle Medium (D-MEM).    -   2. One day before transfection, plate 6×10⁶ cells in 15 ml of        serum supplemented D-MEM per transfection sample in a 10-cm        tissue culture plate. Prepare ten 10-cm plates    -   3. For each 10-cm plate, prepare DNA-Lipofectamine complexes        following steps 4-7.    -   4. Dilute 25 μg of maxi-prep fully human antibody library        plasmid DNA in 1.5 ml Opti-MEM Reduced Serum Medium. Mix gently.    -   5. Dilute 60 μl Lipofecctamine in 1.5 ml Opti-MEM Reduced Serum        Medium. Mix gently and incubate for 5 min at room temperature.    -   6. Combine the diluted DNA with the diluted Lipofectamine. Mix        gently and incubate for 20 min at room temperature.    -   7. Add the 3 ml DNA-Lipofectamine complexes to each plate        containing cells and medium. Mix gently by rocking the plate        back and forth.    -   8. Incubate the cells at 37° C. in a 5% CO₂ incubator overnight.    -   9. Medium change each plate with 15 ml of serum supplemented        D-MEM. Incubate the cells at 37° C. in a 5% CO₂ incubator for 48        hours.    -   10. Detach cells with Detachin cell detachment medium and        re-suspend cells in serum supplemented D-MEM.    -   11. Plate 0.4×10⁶ cells in 10 ml of serum supplemented D-MEM        with 800 μg/ml G418 in one 10-cm tissue culture plate. Transfer        all transfected cells resulting in 150×10-cm plates.    -   12. Plate 0.4×10⁶ un-transfected CHO-S cells in 10 ml of serum        supplemented D-MEM with 800 μg/ml G418 in one 10-cm tissue        culture plate.    -   13. Feed the cells with serum supplemented D-MEM with 800 μg/ml        G418 every 4 days.    -   14. After 14 days, inspect the plates with non-transfected CHO-S        cells. There should be no live cells on the plate.    -   15. Detach the remaining transfected cells with Detachin cell        detachment medium and freeze the cells in freezing media at        1×10⁷ cells/ml.

Appendix 1.4: Generation of Stable Fully Human Antibody Library inMammalian Cells by Retroviral Infection

-   -   1. One day before transfection, plate 6×10⁶ EcoPack-2 293 cells        in 15 ml of serum supplemented D-MEM per transfection sample in        a 10-cm tissue culture plate. Prepare ten 10-cm plates.    -   2. Prepare the MBS-containing medium. This is done immediatelye        prior to the transfection. For each 10-cm tissue culture plate,        12 ml of MBS-containing medium must be prepared.    -   3. Add 12 ml of MBS-containing medium to each 10-cm plate and        return the plates to the plates to the 37° C. incubator. This        must be done 20-30 minutes before the addition of the DNA        suspension.    -   4. Resuspend 10 μg maxi-prep fully human antibody library        plasmid DNA pellet in 450 μl sterile H2O and transfer the DNA to        separate 5-ml BD Falcon polystyrene round-bottom tubes.    -   5. Add 50 μl of Solution I and 500 μ Solution II from the        Stratagene Transfection MBS Mammalian Transfection Kit to the        DNA.    -   6. Gently resuspend any precipitate in the DNA suspension by        pipetting the suspension up and down with a pipettor set at 500        μl.    -   7. Incubate the DNA suspension at room temperature for 10        minutes.    -   8. Remove the 10-cm plates to be transfected from the incubator        and add the DNA suspension onto the plates in a dropwise        fashion, swirling gently to prevent the cells from being lifted        from the plate and to distribute the DNA suspension evenly.    -   9. Return the tissue culture plates to the 37° C. incubator.    -   10. After incubating for 3 hours, remove the medium from the        plates and replace it with 4 ml of 4 ml serum supplemented D-MEM        supplemented with 25 μM chloroquine. Return the plates to the        37° C. incubator.    -   11. After incubating for an additional 6-7 hours, remove the        growth medium containing 25 μM chloroquine and replace with 4 ml        serum supplemented D-MEM without chloroquine.    -   12. Incubate the plate in the 37° C. incubator overnight.    -   13. Remove medium from the plates and replace with 3.0 ml of        fresh serum supplemented D-MEM. Return the plates to the 37° C.        incubator.    -   14. Remove the virus-producing packaging cells from the        incubator.    -   15. Collect the virus-containing supernatant from the first        plate and filter it through a 0.45 pm filter into a sterile        50-ml conical tube.    -   16. Aliquot the viral supernatant into 1.5 ml cryovials and snap        freeze in dry ice/ethanol bath. Store the viral supernatant at        -80° C.    -   17. Plate 0.5×10⁶ NIH-3T3 cells in 10 ml of serum supplemented        D-MEM in one 10-cm tissue culture plate. Plate 102×10-cm tissue        culture plates.    -   18. Quickly thaw the supernatant by rapid agitation in a 37° C.        H2O bath.    -   19. Dilute the virus in calf serum supplemented DMEM with        DEAE-dextran solution at the titer of 0.3×10⁵ viral particle/ml.        Prepare 3 ml diluted virus per100-mm plate to be infected (20%        of the cells will be infected). Prepared “mock cocktail” of        growth medium plus DEAE-dextran to be used as the negative        control.    -   20. Aspirate off medium from NIH-3T3 cells. For each plate,        spread 3.0 ml diluted virus evenly over the cells. Return the        plates to the 37° C. incubator for 3 hours.    -   21. After the 3 hour incubation, add an additional 7.0 ml calf        serum supplemented D-MEM to each plate, and return the plates to        the 37° C. incubator. Incubate the plate for 48 hours.    -   22. Replace medium with 10 ml of calf serum supplemented D-MEM        with 800 μg/ml G418 in each 10-cm tissue culture plate.    -   23. Feed the cells with calf serum supplemented D-MEM with 800        μg/ml G418 every 4 days.    -   24. After 14 days, inspect the plates with mock-infected NIH-3T3        cells. There should be no live cells on the plate.    -   25. Detach the remaining transfected cells with Detachin cell        detachment medium and freeze the cells in freezing media at        1×10⁷ cells/ml.

Example 2 Reactions for Comprehensive Positional Evolution (CPE)

Mutagenesis Reaction

One pair of primers (Primer mix 1 and Primer mix 2) is designed for eachcodon to be inserted. Design will depend on gene sequence, and sequenceanalysis databases such as Sequencher (Gene Codes Corporation) orVectorNTI® (Life Technologies) can be used to design the primers. ForCPE, one pair of primers is designed for each codon to be mutated. Adegenerate target codon (NNK or NNN) is in the middle, flanked by 20bases on each side (total primer length: 43 bases, 9f clones forsequencing to identify unique mutants). Template DNA is vector DNA withtarget gene(s).

Prepare the following reactions in 96-well thin wall PCR plates or 0.2ml thin wall PCR tubes on ice:

Primer mix 1 (2.5 μM) 5 μl Primer mix 2 (2.5 μM) 5 μl 10× Pfu turbo DNApolymerase buffer 2.5 μl DNA template (5, 10, 25 ng) x μl dNTPs 2 μlNuclease-free water QS to 24.5 μl Pfu turbo DNA polymerase (2.5 Uμl) 0.5μl Total reaction volume 25 μl

-   -   1. Prepare one negative control reaction per one 96-well plate        (replace primers with TE buffer)    -   2. Mix gently and spin briefly (5 sec.) in table top centrifuge    -   3. Cycle the reactions using the cycling parameters outlined        below:

Segment Cycles Temperature Time 1  1 95° C. 30 seconds 2 18 95° C. 30seconds 55° C. 1 minute 68° C. 16 minutes

Quality Control Analysis

-   -   1. To QC the amplification reactions, set up the following        reactions in 96-well thin wall PCR plates or 0.2 ml thin wall        PCR tubes:

Mutagenesis reaction 5 μl Water 4 μl Sample loading buffer 1 μl Volume10 μl 

-   -   2. Load 10 μl onto a 1% agarose TAE gel with 0.5 μg/ml Ethidium        Bromide. Use 1 kb plus DNA ladder as standard. Run the gel at        100V for 20-30 minutes in 1× TAE buffer.        Digest the Mutagenesis Reactions with restriction enzymes        appropriate for cloning into vector DNA—Example for DpnI        restriction enzyme    -   1. Add 0.5 μl of the DpnI restriction enzyme (10 U/μl) directly        to each reaction.    -   2. Mix gently and spin briefly (5 sec.) in a table top        centrifuge    -   3. Incubate at 37° C. in PCR machines for 2 hours.    -   4. Transform 6 reaction mixtures from each of 96-well plate into        XLI Blue Supercompetent cells. Store the rest of the reactions        at −20° C.    -   5. Pre-chill 0.2 ml PCR tubes on ice. Warm SOC medium to 42° C.    -   6. Thaw the XLI Blue Supercompetent cells on ice. When thawed,        gently mix and aliquot 50u1 of cells into each of the        pre-chilled tubes.    -   7. Add 0.8 μl of beta-mercaptoethanol to each aliquot of cells.        Incubate the cells on ice for 10 minutes, swirling gently every        2 minutes.    -   8. Add 2 μl of the reaction mixture to one aliquot of cells.        Flick the tubes gently.    -   9. Incubate the tubes on cold blocks for 30 minutes.    -   10. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.    -   11. Incubate the tubes on ice for 2 minutes    -   12. Add 100 μl of preheated SOC medium and incubate the tubes at        37° C. for 1 hour with shaking at 225-250 rpm.    -   13. Plate the entire transformation mixture on LB agar plates        containing carbenicillin.    -   14. Incubate the plates at 37° C. overnight.    -   15. Count colonies on plates and pick 12 colonies from each        transformation reaction for miniprep and sequencing.

Large Scale Transformation

-   -   1. Thaw the XLI Blue Supercompetent cells on ice. Thaw 20 tubes        of competent cells for 96 reactions. When thawed, add 4 μl of        β-mercaptoethanol to each tube of 250 μl competent cells.        Incubate the cells on ice for 10 minutes, swirling gently every        2 minutes.    -   2. Pre-chill 0.2 ml PCR tubes on ice. Warm SOC medium to 42° C.    -   3. Aliquot 50 μl of cells into each of the pre-chilled tubes.    -   4. Add 2 μl of the reaction mixture to one aliquot of cells.        Flick the tubes gently.    -   5. Incubate the tubes on cold blocks for 30 minutes.    -   6. Heat-pulse the tubes in a 42° C. water bath for 45 seconds.    -   7. Incubate the tubes on ice for 2 minutes,    -   8. Add 100 μl of preheated SOC medium and incubate the tubes at        37° C. for 1 hour with shaking at 225-250 rpm.    -   9. Plate the entire transformation mixture on LB agar plates        containing carbenicillin.    -   10. Incubate the plates at 37° C. overnight.    -   11. Grow cells for in 96 well blocks for miniprep    -   12. Prepare miniprep DNA using QIAVac 96 kit following        manufacture's protocol.

Example 3 Screening for Antibody Affinity Improvement

Transfection

One week before transfection, transfer 293F cells to monolayer culturein serum supplemented Dulbecco's Modified Eagle Medium (D-MEM). One daybefore transfection, plate 0.2×10⁵ and 0.4×10⁵ cells in 100 μl of serumsupplemented D-MEM per transfection sample in 96 well formats.

-   -   1. For each transfection sample, prepare DNA-Lipofectamine        complexes.    -   2. Dilute 0.2 μg of DNA in 50 μl Opti-MEM Reduced Serum Medium.        Mix gently.    -   3. Dilute 0.125 μl Lipofecctamine in 50 μl Opti-MEM Reduced        Serum Medium. Mix gently and incubate for 5 mM at room        temperature.    -   4. Combine the diluted DNA with the diluted Lipofectamine Mix        gently and incubate for 20 mM at room temperature.    -   5. Add the 100 μl DNA-Lipofectamine complexes to each well        containing cells and medium. Mix gently by rocking the plate        back and forth.    -   6. Incubate the cells at 37° C. in a 5% CO₂ incubator.    -   7. Add 100 μl of serum supplemented D-MEM to each well after 6        hours. Incubate the cells at 37° C. in a 5% CO₂ incubator        overnight.    -   8. Aspirate off medium in each well. Wash each well with 100 μl        of 293 SFM II with 4 mM L-Glutamine Add 100 μl of 293 SFM II        with 4 mM L-Glutamine to each well.    -   9. Collect supernatant for ELISA at 96 hours after transfection.

Functional ELISA

-   -   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μl of 2        μg/ml antigen in coating solution.    -   2. Cover plates with sealers and incubate overnight at 4C.    -   3. In the event library screening to mimic formulation processes        is desired, one can freeze/dry 100 μl of supernatant from        transfection using 96 well freeze drying systems such as VirTis        96 well Freeze Drying Systems.    -   4. Decant plates and tap out residue liquid, if appropriate.    -   5. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   6. Decant plates and tap out residue liquid.    -   7. Add 200 μl blocking solution. Shake at 200 rpm for 1 hour at        room temperature.    -   8. Decant plates and tap out residue liquid.    -   9. Add duplicates of 100 μl/well of control antibody (2 μg/ml)        in blocking solution to the plates.    -   10. Add duplicates of 100 μl of supernatant from transfection to        the plates.    -   11. Resuspend the dried supernatant in 100 μl of screening        solution and add to the plates.        -   Note: The screening solution may contain the following:            -   Sodium acetate, sodium succinate, gluconate, hisitidine,                citrate or other organic acid to adjust to the pH range                used in the formulation            -   Polyol such as sucrose or trehalose in the range of 1 to                15%            -   Surfactant such as polysorbates or poloxamers in the                range of concentration such 0.001%-0.5% or 0.005% to                0.2% or 0.01% to 0.1%            -   Carriers or stabilizers such as ascorbic acid and                methionine (anti-oxidants), EDTA (chelating agents),                Zn-protein complexes (metal complexes), polymers                (polyesters)            -   Additional salts such as sodium, calcium or Zinc.    -   12. Shake at 200 rpm for one hour at room temperature or other        temperatures which mimic the formulation process.    -   13. Decant plates and tap out residual liquid.    -   14. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   15. Repeat step 11-12 3 times.    -   16. Add 100 μl of 1:5000 dilution of affinity purified goat        anti-human antibody conjugate with HRP in blocking solution to        each well.    -   17. Shake at 200 rpm for one hour at room temperature.    -   18. Decant plates and tap out residual liquid.    -   19. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   20. Repeat step 17-18 3 times.    -   21. Add 100 μl of Sigma TMB substrate to each well. Incubate at        room temperature and check every 2-5 minutes.    -   22. Add 100 μl 1N HCl to stop the reaction.    -   23. Read at 450 nm.

Quantitation ELISA

-   -   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μl of 10        μg/ml affinity-purified Fc-specific goat anti-human IgG in        coating solution.    -   2. Cover plates with sealers and incubate overnight at 4C.    -   3. Decant plates and tap out residue liquid.    -   4. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   5. Decant plates and tap out residue liquid.    -   6. Add 200 μl blocking solution. Shake at 200 rpm for 1 hour at        room temperature.    -   7. Decant plates and tap out residue liquid.    -   8. Add duplicates of 100 μl/well of standardized concentration        of purified human serum IgG in blocking solution to the plates.    -   9. Add duplicates of 100 μl of supernatant from transfection        (SOP 5A) to the plates.    -   10. Shake at 200 rpm for one hour at room temperature.    -   11. Decant plates and tap out residual liquid.    -   12. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   13. Repeat step 11-12 3 times.    -   14. Add 100 μl of 1:5000 dilution of affinity purified goat        anti-human antibody conjugate with HRP in blocking solution to        each well.    -   15. Shake at 200 rpm for one hour at room temperature.    -   16. Decant plates and tap out residual liquid.    -   17. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   18. Repeat step 17-18 3 times.    -   19. Add 100 μl of Sigma TMB substrate to each well. Incubate at        room temperature and check every 2-5 minutes.    -   20. Add 100 μl 1N HCl to stop the reaction.    -   21. Read at 450 nm.

Example 4 Generation and Screening of an Fc Codon Variant Library forOptimal Antibody Expression

The present example provides methods for generating a Fc codon variantlibrary and screening methods for obtaining Fc variants with optimizedfor improved expression in production host cells as compare to theparental form of Fc polypeptide.

A. Design and Construction of a Fc codon variant library

For each codon in the target area (in this case the Fc part of the humanIgGl molecule) a pair of degenerate primers (forward and reverse) isdesigned that includes the target codon and 20 bases on each side. The3^(rd) position of the target codon (wobble position) contains mixedbases (Table 3) that allow the generation of all silent mutations at thetarget position using the same codon (example A). A second set ofdegenerate primer is designed for the same codon position if thecorresponding amino acid can be encoded by another codon (example B).Corresponding forward and reverse degenerate primers are mixed 1:1,annealed to the template and extended to full length products by stranddisplacement using a thermostable DNA polymerase. Template is digestedwith Dpnl and full length extension products are transformed into E.coli. Up to 12 colonies per mutagenesis reaction are sequenced. Sequenceconfirmed mutants are arrayed in 96 well plates and glycerol stocked.The glycerol stocks are used to miniprep plasmid DNA for transfectioninto mammalian cells and screening.

TABLE 3 Codes for degenerate bases in synthetic oligos Mixed Symbol BaseR A, G Y C, T M A, C K G, T S C, G W A, T H A, C, T B C, G, T V A, C, GD A, G, T N A, C, G, T

Example A: target codon=CCC (proline)

→forward primer: CCD, reverse primer: HGG

Example B; target codon=TCG (serine)

→forward primerl: TCH, reverse primerl: DGA

→forward primer2: AGY, reverse primer2: RCT

-   -   20 bases flanking the target codon are not shown. Total primer        length: 43 bases.

B. Expression and ELISA based screening of Fc codon variant library

Clones from the Fc codon variant library were transfected into amammalian cell line. Full length IgGs were produced and secreted intothe medium. Supernatants of expressed Fc codon variants were screenedfor IgG expression level higher than the parental clone using ELISAassay. The ELISA data was normalized with beta-galactosidase assaymeasuring the transfection efficiency. Top hits identified in theprimary screen were re-transfected and re-screened three times toconfirm the increased expression level.

Example 5 Method of Screening the Antibody Clones in the Conditions thatMimic the Formulation Process and in the Presence of FormulationComponents Day 1

-   -   1. Coat Nunc-Immuno Maxisorp 96 well plates with 100 μl of 2        μg/ml antigen in coating solution.    -   2. Cover plates with sealers and incubate overnight at 4C.    -   3. Freeze/dry 100 μl of supernatant from transfection (SOPSA or        SOP9A) using 96 well freeze drying systems such as VirTis 96        wellFreeze Drying Systems

Day 2

-   -   4. Decant plates and tap out residue liquid.    -   5. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   6. Decant plates and tap out residue liquid.    -   7. Add 200 μl blocking solution. Shake at 200 rpm for 1 hour at        room temperature.    -   8. Decant plates and tap out residue liquid.    -   9. Resuspend the dried supernatant in 100 μl of screening        solution and add to the plates.        -   Note: The screening solution may contain the following:            -   1. Sodium acetate, sodium succinate, gluconate,                hisitidine, citrate or other organic acid to adjust to                the pH range used in the formulation            -   2. Polyol such as sucrose or trehalose in the range of 1                to 15%            -   3. Surfactant such as polysorbates or poloxamers in the                range of concentration such 0.001%-0.5% or 0.005% to                0.2% or 0.01% to 0.1%            -   4. Carriers or stabilizers such as ascorbic acid and                methionine (anti-oxidants), EDTA (chelating agents),                Zn-protein complexes (metal complexes), polymers                (polyesters)            -   5. Additional salts such as sodium, calcium or Zinc    -   10. Shake at 200 rpm for one hour at room temperature or other        temperatures which mimic the formulation process    -   11. Decant plates and tap out residual liquid.    -   12. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   13. Repeat step 11-12 3 times.    -   14. Add 100 μl of 1:5000 dilution of affinity purified goat        anti-human antibody conjugate with HRP in blocking solution to        each well.    -   15. Shake at 200 rpm for one hour at room temperature.    -   16. Decant plates and tap out residual liquid.    -   17. Add 200 μl washing solution. Shake at 200 rpm for 5 min at        room temperature.    -   18. Repeat step 17-18 3 times.    -   19. Add 100 μl of Sigma TMB substrate to each well. Incubate at        room temperature and check every 2-5 minutes.    -   20. Add 100 μl 1N HCl to stop the reaction.    -   21. Read at 450 nm.

Washing solution

-   -   0.05% Tween-20 in PBS

Screening solution

-   -   2% Carnation non-fat milk in PBS and may also include the        following        -   Sodium acetate, sodium succinate, gluconate, hisitidine,            citrate or other organic acid to adjust to the pH range used            in the formulation        -   Polyol such as sucrose or trehalose in the range of 1 to 15%        -   Surfactant such as polysorbates or poloxamers in the range            of concentration such 0.001%-0.5% or 0.005% to 0.2% or 0.01%            to 0.1%        -   Carriers or stabilizers such as ascorbic acid and methionine            (anti-oxidants), EDTA (chelating agents), Zn-protein            complexes (metal complexes), polymers (polyesters)        -   Additional salts such as sodium, calcium or Zinc

Incorporation of Material of ASCII Text Sequence Listing by Reference

The material in the ASCII text file sequence listing named,“BIAT1006USCIPC2_Sequence_Listing” created on Mar. 27, 2021, which is 1kb in size, is hereby incorporated by reference in its entirety herein.

1-75. (canceled)
 76. A method of evolution of a template human proteinin a manufacturing host, the method comprising steps of: a. mutating thetemplate human protein to produce a set of mutant human proteins in themanufacturing host, wherein in this mutating step the manufacturing hostincorporates an unnatural amino acid into the mutant human proteins; andb. screening the set of mutant human proteins produced in step (a) forat least one predetermined property, characteristic or activity.
 77. Themethod of claim 76 further comprising a step of: c. selecting anup-mutant human protein from the set of mutant human proteins screenedin step (b) based upon the at least one predetermined property,characteristic or activity.
 78. The method of claim 77, the methodfurther comprising a step of: d. manufacturing the up-mutant humanprotein selected in step (c) by expressing the up-mutant human proteinin the same manufacturing host as was used in the mutating step (a). 79.The method of claim 77, wherein the selecting step (c) comprisesselecting an up-mutant human protein from the set of mutant humanproteins based upon:, (1) optimization of the at least one predeterminedproperty, characteristic or activity when compared to the template humanprotein, and (2) modified expression when compared to the template humanprotein.
 80. The method of claim 79 wherein the modified expression isenhanced expression. 81-83. (canceled)
 84. The method of claim 76,wherein the screening step (b) is carried out under screening conditionsthat mimic manufacturing conditions.
 85. The method of claim 76, whereinthe screening step (b) is carried out under cell free conditions. 86.The method of claim 76, wherein the screening step (b) is carried out ina non-human living organism.
 87. The method of claim 76, wherein thescreening step (b) identifies mutant human proteins exhibiting anundesirable immune response and further comprising a step of: (c)selecting the mutant proteins that do not exhibit the undesirable immuneresponse.
 88. The method of claim 76, wherein the template human proteinis an antibody.
 89. The method of claim 88, wherein the antibody is afull length antibody.
 90. The method of claim 89, wherein the at leastone predetermined property, characteristic or activity in screening step(b) comprises one or more of: (1) screening for a silent mutation and(2) screening for a missense mutation; each compared to the templateantibody.
 91. The method of claim 76, wherein the human protein isselected from the group consisting of an enzyme, a cytokine, a receptor,a DNA binding protein, a chelating agent and a hormone.
 92. The methodof claim 77, wherein the manufacturing host is a eukaryotic cellproduction host and the evolving step comprises evolving the templatehuman protein to produce a set of mutant human proteins in theeukaryotic cell production host with cell surface display.
 93. Themethod of claim 92, wherein the up-mutant human protein comprises atleast one silent mutation and/or missense mutation.
 94. A method ofevolution of a template human protein in a manufacturing host, themethod comprising steps of: a. mutating the template human protein toproduce a set of mutant human proteins in the manufacturing host,wherein in this mutating step the manufacturing host reduces fucosecontent in the mutant human proteins relative to the template humanprotein; b. screening the set of mutant human proteins produced in step(a) for at least one predetermined property, characteristic or activity;and c. selecting an up-mutant human protein from the set of mutant humanproteins screened in step (b) based upon the at least one predeterminedproperty, characteristic or activity.
 95. The method of claim 94, themethod further comprising a step of: d. manufacturing the up-mutanthuman protein selected in step (c) by expressing the up-mutant humanprotein in the same manufacturing host as was used in the mutating step(a).
 96. The method of claim 95, wherein the selecting step (c)comprises selecting an up-mutant human protein from the set of mutanthuman proteins based upon: (1) optimization of the at least onepredetermined property, characteristic or activity when compared to thetemplate human protein, and (2) enhanced expression when compared to thetemplate human protein.
 97. The method of claim 94, wherein thescreening step (b) identifies mutant human proteins exhibiting anundesirable immune response and further comprising a step of: (c)selecting the mutant proteins that do not exhibit the undesirable immuneresponse.
 98. The method of claim 94, wherein the template human proteinis an antibody.